Nucleic acid adjuvants

ABSTRACT

Recombinant nucleic acid molecules are described. The molecules have two nucleic acid sequences, wherein the first nucleic acid sequence is a truncated A subunit coding region obtained or derived from a bacterial ADP-ribosylating exotoxin, and the second nucleic acid sequence is a truncated B subunit coding region. Vectors and compositions containing these molecules are also described. Methods for enhancing an immune response against an antigen of interest using these recombinant nucleic acid molecules and compositions are also described.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is related to U.S. provisional applicationserial No. 60/253,381, filed Nov. 27, 2000, from which priority isclaimed pursuant to 35 U.S.C. §119(e)(1) and which application isincorporated herein by reference in its entirety.

TECHNICAL FIELD

[0002] The invention relates to the fields of molecular biology andimmunology, and generally relates to nucleic acid immunizationtechniques. More specifically, the invention relates to polynucleotidesencoding an adjuvant, and to immunization strategies employing suchpolynucleotides.

BACKGROUND

[0003] Techniques for the injection of DNA and mRNA into mammaliantissue for the purposes of immunization against an expression producthave been described in the art. The techniques, termed “nucleic acidimmunization” herein, have been shown to elicit both humoral andcell-mediated immune responses. For example, sera from mice immunizedwith a DNA construct encoding the envelope glycoprotein, gp160, wereshown to react with recombinant gp160 in immunoassays, and lymphocytesfrom the injected mice were shown to proliferate in response torecombinant gp120. Wang et al. (1993) Proc. Natl. Acad. Sci. USA90:4156-4160. Similarly, mice immunized with a human growth hormone(hGH) gene demonstrated an antibody-based immune response. Tang et al.(1992) Nature 356:152-154. Intramuscular injection of DNA encodinginfluenza nucleoprotein driven by a mammalian promoter has been shown toelicit a CD8+CTL response that can protect mice against subsequentlethal challenge with virus. Ulmer et al. (1993) Science 259:1745-1749.Immunohistochemical studies of the injection site revealed that the DNAwas taken up by myeloblasts, and cytoplasmic production of viral proteincould be demonstrated for at least 6 months.

SUMMARY OF THE INVENTION

[0004] It is a primary object of the invention to provide apolynucleotide adjuvant composition containing first and second nucleicacid sequences, wherein the first nucleic acid sequence is a truncated Asubunit coding region obtained or derived from a bacterialADP-ribosylating exotoxin, and the second nucleic acid sequence is atruncated B subunit coding region obtained or derived from a bacterialADP-ribosylating exotoxin. Each of the truncated subunit coding regionshas a 5′ deletion and encodes a subunit peptide not having an aminoterminal bacterial signal peptide.

[0005] The first and second nucleic acid sequences may be present in thesame or in different nucleic acid constructs. The truncated subunitcoding regions may be obtained or derived from the same bacterialADP-ribosylating exotoxin and, in certain preferred embodiments, thebacterial ADP-ribosylating exotoxin is a cholera toxin (CT) or an E.coli heat labile enterotoxin (LT). In addition, at least one of thetruncated subunit coding regions may be genetically modified to detoxifythe subunit peptide encoded thereby, for example wherein the truncated Asubunit coding region has been genetically modified to disrupt orinactivate ADP-ribosyl transferase activity in the subunit peptideexpression product.

[0006] It is also a primary object of the invention to provide apolynucleotide adjuvant composition containing first and second nucleicacid sequences, wherein the first nucleic acid sequence is a modified Asubunit coding region obtained or derived from a bacterialADP-ribosylating exotoxin, and the second nucleic acid sequence is a Bsubunit coding region obtained or derived from a bacterialADP-ribosylating exotoxin. The modified A subunit coding region and saidB subunit coding region each encode a mature subunit peptide, and themodified A subunit coding region has been genetically modified so as todelete a C-terminal KDEL or RDEL motif in the subunit peptide encodedthereby.

[0007] As above, the first and second nucleic acid sequences may bepresent in the same or in different nucleic acid constructs. Thetruncated subunit coding regions may be obtained or derived from thesame bacterial ADP-ribosylating exotoxin and, in certain preferredembodiments, the bacterial ADP-ribosylating exotoxin is a cholera toxin(CT) or an E. coli heat labile enterotoxin (LT). In addition, at leastone of the truncated subunit coding regions may be genetically modifiedto detoxify the subunit peptide encoded thereby, for example wherein thetruncated A subunit coding region has been genetically modified todisrupt or inactivate ADP-ribosyl transferase activity in the subunitpeptide expression product.

[0008] In certain aspects of the invention, the above compositions canbe provided in particulate form, for example wherein the compositionsare particulates suitable for delivery from a particle delivery device.In this regard, the present compositions may be coated onto the same ora different core carrier particle and thus suitable for delivery using aparticle-mediated transfection technique. Preferred core carrierparticles will have an average diameter of about 0.1 to 10 μm, and maycomprise a metal such as gold. Accordingly, it is a still further objectof the invention to provide a particle delivery device loaded with(e.g., containing) a particulate composition as defined herein.

[0009] It is also a primary object of the invention to provide a methodfor enhancing an immune response against an antigen of interest in asubject. The method generally entails: (a) administering the antigen ofinterest to the subject; (b) providing an adjuvant compositioncomprising first and second nucleic acid sequences, wherein the firstnucleic acid sequence is a truncated A subunit coding region obtained orderived from a bacterial ADP-ribosylating exotoxin, and the secondnucleic acid sequence is a truncated B subunit coding region obtained orderived from a bacterial ADP-ribosylating exotoxin; and (c)administering the adjuvant composition to the subject, whereby uponintroduction to the subject, the first and second nucleic acid sequencesare expressed to provide subunit peptides in an amount sufficient toelicit an enhanced immune response against the antigen of interest. Thesubunit coding regions are truncated in that each coding region has a 5′deletion and encodes a subunit peptide not having an amino terminalbacterial signal peptide.

[0010] It is yet a further primary object of the invention to provide amethod for enhancing an immune response against an antigen of interestin a subject, wherein the method entails: (a) administering the antigenof interest to the subject; (b) providing an adjuvant compositioncomprising first and second nucleic acid sequences, wherein the firstnucleic acid sequence is a modified A subunit coding region obtained orderived from a bacterial ADP-ribosylating exotoxin, and the secondnucleic acid sequence is a B subunit coding region obtained or derivedfrom a bacterial ADP-ribosylating exotoxin; and (c) administering theadjuvant composition to the subject, whereby upon introduction to thesubject, the first and second nucleic acid sequences are expressed toprovide subunit peptides in an amount sufficient to elicit an enhancedimmune response against the antigen of interest. The subunit codingregions are modified in that each encodes a mature subunit peptide, butthe A subunit coding region has been genetically modified so as todelete a C-terminal KDEL or RDEL motif in the subunit peptide encodedthereby.

[0011] In the methods of the invention, administering the adjuvantcompositions entails transfecting cells of the subject with apolynucleotide adjuvant composition according to the present invention.Expression cassettes and/or vectors containing the nucleic acidmolecules of the present invention can be used to transfect the cells,and transfection is carried out under conditions that permit expressionof the subunit peptides within the subject. The method may furtherentail one or more steps of administering at least one secondarycomposition to the subject.

[0012] The transfection procedure carried out during the immunizationcan be conducted either in vivo, or ex vivo (e.g., to obtain transfectedcells which are subsequently introduced into the subject prior tocarrying out the secondary immunization step). When in vivo transfectionis used, the nucleic acid molecules can be administered to the subjectby way of intramuscular or intradermal injection of plasmid DNA or,preferably, administered to the subject using a particle-mediateddelivery technique. Vaccine compositions (containing the antigen ofinterest) can be provided in the form of any suitable vaccinecomposition, for example, in the form of a peptide subunit composition,in the form of a nucleic acid vaccine composition, or in the form of awhole or split virus influenza vaccine composition.

[0013] In certain methods, the antigen of interest and the adjuvantcomposition are administered to the same site in the subject. Forexample, the adjuvant composition and the antigen of interest can beadministered concurrently (e.g., provided in a single vaccinecomposition). In certain preferred embodiments, the adjuvant and,optionally the antigen of interest, is administered in particulate form,for example wherein the adjuvant composition has been coated onto a corecarrier particle and delivered to the subject using a particle-mediateddelivery technique.

[0014] In these methods, administration of the polynucleotide adjuvantcompositions of the present invention preferably results in an augmentedcellular immune response against the co-administered antigen ofinterest. Such an enhanced immune response may be generallycharacterized by increased titers of interferon-producing CD4⁺ and/orCD8⁺ T lymphocytes, increased antigen-specific cytotoxic T lymphocyte(CTL) activity, and a T helper 1-like immune response (Th1) against theantigen of interest (characterized by increased antigen-specificantibody titers of the subclasses typically associated with cellularimmunity (e.g., IgG2a), usually with a concomitant reduction of antibodytiters of the subclasses typically associated with humoral immunity(e.g., IgG1)) instead of a T helper 2-like immune response (Th2) such asthat normally produced when immunizing a subject using a bacterialADP-ribosylating exotoxin adjuvant such as CT or LT.

[0015] Advantages of the present invention include, but are not limitedto the ability of the present adjuvant compositions to providesignificant adjuvant effect and thereby enhance the immunogenicity of aco-administered antigen in an immunized subject, as well as the abilityto favor a Th1-like immune response against the co-administered antigenwhich is beneficial in a vaccine product.

[0016] These and other objects, aspects, embodiments and advantages ofthe present invention will readily occur to those of ordinary skill inthe art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1 is a restriction map and functional map of plasmid pPJV2002that contains a truncated coding sequence for a Cholera Toxin (CT)subunit A (CTA) peptide, wherein the plasmid further contains the humancytomegalovirus (hCMV) immediate early promoter and associated intron Asequence, and the coding sequence for the signal peptide of human tissueplasminogen activator to allow for secretion from mammalian cells of thetruncated CTA expression product. The figure further contains thecomplete nucleic acid sequence (SEQ ID NO: 1) for the pPJV2002 plasmid.

[0018]FIG. 2 is a restriction map and functional map of plasmid pPJV2003that contains a truncated coding sequence for a Cholera Toxin (CT)subunit B (CTB) peptide, wherein the plasmid further contains the hCMVimmediate early promoter and associated intron A sequence, and thecoding sequence for the signal peptide of human tissue plasminogenactivator to allow for secretion from mammalian cells of the truncatedCTB expression product. The figure further contains the complete nucleicacid sequence (SEQ ID NO: 2) for the pPJV2003 plasmid.

[0019]FIG. 3 is a restriction map and functional map of plasmid pPJV2006that contains a truncated coding sequence for a CTA peptide, wherein thetruncated CTA coding sequence has been further modified to delete aC-terminal KDEL motif in the subunit peptide encoded thereby. Theplasmid further contains the hCMV immediate early promoter andassociated intron A sequence, and the coding sequence for the signalpeptide of human tissue plasminogen activator to allow for secretionfrom mammalian cells of the truncated CTA expression product. The figurefurther contains the complete nucleic acid sequence (SEQ ID NO: 3) forthe pPJV2006 plasmid.

[0020]FIG. 4 is a restriction map and functional map of plasmid pPJV2004that contains a truncated coding sequence for an E. coli heat labileenterotoxin (LT) subunit A (LTA) peptide, wherein the plasmid furthercontains the hCMV immediate early promoter and associated intron Asequence, and the coding sequence for the signal peptide of human tissueplasminogen activator to allow for secretion from mammalian cells of thetruncated LTA expression product. The figure further contains thecomplete nucleic acid sequence (SEQ ID NO: 4) for the pPJV2004 plasmid.

[0021]FIG. 5 is a restriction map and functional map of plasmid pPJV2005that contains a truncated coding sequence for an LT subunit B (LTB)peptide, wherein the plasmid further contains the hCMV immediate earlypromoter and associated intron A sequence, and the coding sequence forthe signal peptide of human tissue plasminogen activator to allow forsecretion from mammalian cells of the truncated LTB expression product.The figure further contains the complete nucleic acid sequence (SEQ IDNO: 5) for the pPJV2005 plasmid.

[0022]FIG. 6 is a restriction map and functional map of plasmid pPJV2007that contains a truncated coding sequence for an LTA peptide, whereinthe truncated LTA coding sequence has been further modified to delete aC-terminal RDEL motif in the subunit peptide encoded thereby. Theplasmid further contains the hCMV immediate early promoter andassociated intron A sequence, and the coding sequence for the signalpeptide of human tissue plasminogen activator to allow for secretionfrom mammalian cells of the truncated LTA expression product. The figurefurther contains the complete nucleic acid sequence (SEQ ID NO: 6) forthe pPJV2007 plasmid.

[0023]FIG. 7 depicts the results from the ELISA carried out in Example5. The histogram represents the log reciprocal titer of anti-gp120antibody present in the animals receiving, from left to right in thefigure, Formulation #1 containing the empty pWRG7054 vector (“EmpVec”);Formulation #2 containing the EmpVec (pWRG7054) and the pCIA-EnvTplasmid (“gp120”); Formulation #3 containing the EmpVec (pWRG7054)combined with the pPJV2002 and pPJV2003 adjuvant vectors (“CTA/B”);Formulation #4 containing the pCIA-EnvT plasmid (“gp120”) combined withthe pPJV2002 and pPJV2003 adjuvant vectors (“CTA/B”); Formulation #5containing the pCIA-EnvT plasmid (“gp120”) combined with the pPJV2006and pPJV2003 adjuvant vectors (“CTA-KDEL/B”); or no vaccine and/oradjuvant composition (“naive”).

[0024]FIG. 8 depicts the results from the in situ ELISA carried out inExample 5. The histogram represents the relative level of gp120-specificIFN-γ production in splenocytes obtained from animals receiving, fromleft to right in the figure, Formulation #1 containing the emptypWRG7054 vector (“EmpVec”); Formulation #2 containing the EmpVec(pWRG7054) and the pCIA-EnvT plasmid (“gp120”); Formulation #3containing the EmpVec (pWRG7054) combined with the pPJV2002 and pPJV2003adjuvant vectors (“CTA/B”); Formulation #4 containing the pCIA-EnvTplasmid (“gp120”) combined with the pPJV2002 and pPJV2003 adjuvantvectors (“CTA/B”); or Formulation #5 containing the pCIA-EnvT plasmid(“gp120”) combined with the pPJV2006 and pPJV2003 adjuvant vectors(“CTA-KDEL/B”).

[0025]FIG. 9 depicts the results from the ELISPOT assay carried out inExample 5. The histogram represents the relative levels ofIFN-γ-producing splenocytes obtained from animals receiving, from leftto right in the figure, Formulation #1 containing the empty pWRG7054vector (“EmpVec”); Formulation #2 containing the EmpVec (pWRG7054) andthe pCIA-EnvT plasmid (“gp120”); Formulation #3 containing the EmpVec(pWRG7054) combined with the pPJV2002 and pPJV2003 adjuvant vectors(“CTA/B”); Formulation #4 containing the pCIA-EnvT plasmid (“gp120”)combined with the pPJV2002 and pPJV2003 adjuvant vectors (“CTA/B”); orFormulation #5 containing the pCIA-EnvT plasmid (“gp120”) combined withthe pPJV2006 and pPJV2003 adjuvant vectors (“CTA-KDEL/B”).

[0026]FIG. 10 depicts the results from the ELISA carried out in Example6. In this figure, the geometric mean absorbance values represent thetiter of anti-HBcAg antibody present in serum samples (at four differentdilutions) taken at the booster immunization (week 6 of the study) fromanimals receiving either Formulation #1 containing the HBcAg/HBsAgvector plasmid (pWRG7193); or Formulation #2 containing the HBcAg/HBsAgvector plasmid (pWRG7193) combined with the pPJV2002 and pPJV2003adjuvant vectors (“CTA/B”).

[0027]FIG. 11 depicts the results from the ELISA carried out in Example6. In this figure, the geometric mean absorbance values represent thetiter of anti-HBcAg antibody present in serum samples (at four differentdilutions) taken 2 weeks following the booster immunization (week 8 ofthe study) from animals receiving either Formulation #1 containing theHBcAg/HBsAg vector plasmid (pWRG7193); or Formulation #2 containing theHBcAg/HBsAg vector plasmid (pWRG7193) combined with the pPJV2002 andpPJV2003 adjuvant vectors (“CTA/B”).

[0028]FIG. 12 depicts the results from the ELISA carried out in Example7. The histrogram represents the log IgG1::IgG2a ratios from eachimmunization group receiving, from left to right in the figure,Formulation #1 containing the pM2-FL plasmid (“M2”)combined with theempty vector plasmid control (pWRG7054); Formulation #2 containing thepM2-FL plasmid combined with the pPJV2002 and pPJV2003 CTA/B adjuvantvectors (“M2+CT”); or Formulation #7 containing the pM2-FL plasmidcombined with the pPJV2004 and pPJV2005 LTA/B adjuvant vectors(“M2+LT”).

[0029] FIGS. 13A-13D depict the results from the IFN-γ and the IL4ELISPOT assays used to assess the immune response to the Hepatitis Bvirus surface and core antigens in the first study of Example 8. Thehistograms represent the number of spots per 1×10⁵ spleen cells from thevarious experimental groups.

[0030]FIG. 14 depicts the survival results from the HSV-2 viruschallenge study carried out in Example 9.

DETAILED DESCRIPTION OF THE INVENTION

[0031] Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particularlyexemplified molecules or process parameters as such may, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments of the inventiononly, and is not intended to be limiting. In addition, the practice ofthe present invention will employ, unless otherwise indicated,conventional methods of virology, microbiology, molecular biology,recombinant DNA techniques and immunology all of which are within theordinary skill of the art. Such techniques are explained fully in theliterature. See, e.g., Sambrook, et al., Molecular Cloning: A LaboratoryManual (2nd Edition, 1989); DNA Cloning: A Practical Approach, vol. I &II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); APractical Guide to Molecular Cloning (1984); and Fundamental Virology,2nd Edition, vol. I & II (B. N. Fields and D. M. Knipe, eds.).

[0032] All publications, patents and patent applications cited herein,whether supra or infra, are hereby incorporated by reference in theirentirety.

[0033] It must be noted that, as used in this specification and theappended claims, the singular forms “a,” “an” and “the” include pluralreferents unless the content clearly dictates otherwise.

[0034] Definitions

[0035] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although a number ofmethods and materials similar or equivalent to those described hereincan be used in the practice of the present invention, the preferredmaterials and methods are described herein.

[0036] In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

[0037] The term “adjuvant” intends any material or composition capableof specifically or non-specifically altering, enhancing, directing,redirecting, potentiating or initiating an antigen-specific immuneresponse. Thus, coadministration of an adjuvant with an antigen mayresult in a lower dose or fewer doses of antigen being necessary toachieve a desired immune response in the subject to which the antigen isadministered, or coadministration may result in a qualitatively and/orquantitatively different immune response in the subject. Theeffectiveness of an adjuvant can be determined by administering theadjuvant with a vaccine composition in parallel with a vaccinecomposition alone to animals and comparing antibody and/orcellular-mediated immunity in the two groups using standard assays suchas radioimmunoassay, ELISAs, CTL assays, and the like, all well known inthe art. Typically, in a vaccine composition, the adjuvant is a separatemoiety from the antigen, although a single molecule can have bothadjuvant and antigen properties.

[0038] An “adjuvant composition” intends any pharmaceutical compositioncontaining an adjuvant. Adjuvant compositions can be delivered in themethods of the invention while in any suitable pharmaceutical form, forexample, as a liquid, powder, cream, lotion, emulsion, gel or the like.However, preferred adjuvant compositions will be in particulate form. Itis intended, although not always explicitly stated, that moleculeshaving similar biological activity as wild-type or purified peptide orchemical adjuvants.

[0039] The term “peptide” is used in it broadest sense to refer to acompound of two or more subunit amino acids, amino acid analogs, orother peptidomimetics. The subunits may be linked by peptide bonds or byother bonds, for example ester, ether, etc. As used herein, the term“amino acid” refers to either natural and/or unnatural or syntheticamino acids, including glycine and both the D or L optical isomers, andamino acid analogs and peptidomimetics. A peptide of three or more aminoacids is commonly called an “oligopeptide” if the peptide chain isshort. If the peptide chain is long, the peptide is typically referredto as a “polypeptide” or a “protein”.

[0040] An “antigen” refers to any agent, generally a macromolecule,which can elicit an immunological response in an individual. The termmay be used to refer to an individual macromolecule or to a homogeneousor heterogeneous population of antigenic macromolecules. As used herein,“antigen” is generally used to refer to a peptide or carbohydratemolecule that contains one or more epitopes. For purposes of the presentinvention, antigens can be obtained or derived from any appropriatesource. Furthermore, for purposes of the present invention, an “antigen”includes a peptide having modifications, such as deletions, additionsand substitutions (generally conservative in nature) to the nativesequence, so long as the peptide maintains sufficient immunogenicity.These modifications may be deliberate, for example through site-directedmutagenesis, or may be accidental, such as through mutations of hostswhich produce the antigens.

[0041] An “immune response” against an antigen of interest is thedevelopment in an individual of a humoral and/or a cellular immuneresponse to that antigen. For purposes of the present invention, a“humoral immune response” refers to an immune response mediated byantibody molecules, while a “cellular immune response” is one mediatedby T-lymphocytes and/or other white blood cells.

[0042] The term “nucleic acid immunization” is used herein to refer tothe introduction of a nucleic acid molecule encoding one or moreselected antigens into a host cell for the in vivo expression of theantigen or antigens. The term also encompasses introduction of a nucleicacid molecule encoding one or more selected adjuvants into a host cellfor the in vivo expression of the adjuvant or adjuvants. The nucleicacid molecule can be introduced directly into the recipient subject,such as by standard intramuscular or intradermal injection; transdermalparticle delivery; inhalation; topically, or by oral, intranasal ormucosal modes of administration. The molecule alternatively can beintroduced ex vivo into cells which have been removed from a subject. Inthis latter case, cells containing the nucleic acid molecule of interestare re-introduced into the subject such that an immune response can bemounted against the antigen encoded by the nucleic acid molecule, orsuch that the adjuvant encoded by the nucleic acid molecule can exertits adjuvant effect.

[0043] The terms “nucleic acid molecule” and “polynucleotide” are usedinterchangeably herein and refer to a polymeric form of nucleotides ofany length, either deoxyribonucleotides or ribonucleotides, or analogsthereof. Polynucleotides may have any three-dimensional structure, andmay perform any function, known or unknown. Non-limiting examples ofpolynucleotides include a gene, a gene fragment, exons, introns,messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers.

[0044] A polynucleotide is typically composed of a specific sequence offour nucleotide bases: adenine (A); cytosine (C); guanine (G); andthymine (T) (uracil (U) for thymine (T) when the polynucleotide is RNA).Thus, the term nucleic acid sequence is the alphabetical representationof a polynucleotide molecule. This alphabetical representation can beinput into databases in a computer having a central processing unit andused for bioinformatics applications such as functional genomics andhomology searching.

[0045] A “vector” is capable of transferring nucleic acid sequences totarget cells (e.g., viral vectors, non-viral vectors, particulatecarriers, and liposomes). Typically, “vector construct,” “expressionvector,” and “gene transfer vector,” mean any nucleic acid constructcapable of directing the expression of a gene of interest and which cantransfer gene sequences to target cells. Thus, the term includes cloningand expression vehicles, as well as viral vectors. A “plasmid” is avector in the form of an extrachromosomal genetic element.

[0046] A nucleic acid sequence which “encodes” a selected adjuvantand/or antigen is a nucleic acid molecule which is transcribed (in thecase of DNA) and translated (in the case of mRNA) into a polypeptide invivo when placed under the control of appropriate regulatory sequences.The boundaries of the coding sequence are determined by a start codon atthe 5′ (amino) terminus and a translation stop codon at the 3′ (carboxy)terminus. For the purposes of the invention, such nucleic acid sequencescan include, but are not limited to, cDNA from viral, procaryotic oreucaryotic mRNA, genomic sequences from viral or procaryotic DNA or RNA,and even synthetic DNA sequences. A transcription termination sequencemay be located 3′ to the coding sequence.

[0047] A “promoter” is a nucleotide sequence which initiates andregulates transcription of a polypeptide-encoding polynucleotide.Promoters can include inducible promoters (where expression of apolynucleotide sequence operably linked to the promoter is induced by ananalyte, cofactor, regulatory protein, etc.), repressible promoters(where expression of a polynucleotide sequence operably linked to thepromoter is repressed by an analyte, cofactor, regulatory protein,etc.), and constitutive promoters. It is intended that the term“promoter” or “control element” includes full-length promoter regionsand functional (e.g., controls transcription or translation) segments ofthese regions.

[0048] “Operably linked” refers to an arrangement of elements whereinthe components so described are configured so as to perform their usualfunction. Thus, a given promoter operably linked to a nucleic acidsequence is capable of effecting the expression of that sequence whenthe proper enzymes are present. The promoter need not be contiguous withthe sequence, so long as it functions to direct the expression thereof.Thus, for example, intervening untranslated yet transcribed sequencescan be present between the promoter sequence and the nucleic acidsequence and the promoter sequence can still be considered “operablylinked” to the coding sequence.

[0049] “Recombinant” is used herein to describe a nucleic acid molecule(polynucleotide) of genomic, cDNA, semisynthetic, or synthetic originwhich, by virtue of its origin or manipulation is not associated withall or a portion of the polynucleotide with which it is associated innature and/or is linked to a polynucleotide other than that to which itis linked in nature. Two nucleic acid sequences which are containedwithin a single recombinant nucleic acid molecule are “heterologous”relative to each other when they are not normally associated with eachother in nature.

[0050] An “isolated polynucleotide” is a nucleic acid molecule separateand discrete from the whole organism with which the molecule is found innature; or a nucleic acid molecule devoid, in whole or part, ofsequences normally associated with it in nature; or a sequence, as itexists in nature, but having heterologous sequences (as defined below)in association therewith. A sequence is “derived or obtained from” amolecule if it has the same or substantially the same base pair sequenceas a region of the source molecule, its cDNA, complements thereof, or ifit displays sequence identity as described below.

[0051] Techniques for determining nucleic acid and amino acid “sequenceidentity” or “sequence homology” also are known in the art. Typically,such techniques include determining the nucleotide sequence of the mRNAfor a gene and/or determining the amino acid sequence encoded thereby,and comparing these sequences to a second nucleotide or amino acidsequence. In general, “identity” refers to an exactnucleotide-to-nucleotide or amino acid-to-amino acid correspondence oftwo polynucleotides or polypeptide sequences, respectively. Two or moresequences (polynucleotide or amino acid) can be compared by determiningtheir “percent identity.” The percent identity of two sequences, whethernucleic acid or amino acid sequences, is the number of exact matchesbetween two aligned sequences divided by the length of the shortersequences and multiplied by 100. An approximate alignment for nucleicacid sequences is provided by the local homology algorithm of Smith andWaterman, Advances in Applied Mathematics 2:482-489 (1981). Thisalgorithm can be applied to amino acid sequences by using the scoringmatrix developed by Dayhoff, Atlas of Protein Sequences and Structure,M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical ResearchFoundation, Washington, D.C., USA, and normalized by Gribskov, Nucl.Acids Res. 14(6):6745-6763 (1986). An exemplary implementation of thisalgorithm to determine percent identity of a sequence is provided by theGenetics Computer Group (Madison, Wis.) in the “BestFit” utilityapplication. The default parameters for this method are described in theWisconsin Sequence Analysis Package Program Manual, Version 8 (1995)(available from Genetics Computer Group, Madison, Wis.). A preferredmethod of establishing percent identity in the context of the presentinvention is to use the MPSRCH package of programs copyrighted by theUniversity of Edinburgh, developed by John F. Collins and Shane S.Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View,Calif.). From this suite of packages the Smith-Waterman algorithm can beemployed where default parameters are used for the scoring table (forexample, gap open penalty of 12, gap extension penalty of one, and a gapof six). From the data generated the “Match” value reflects “sequenceidentity.” Other suitable programs for calculating the percent identityor similarity between sequences are generally known in the art, forexample, another alignment program is BLAST, used with defaultparameters. For example, BLASTN and BLASTP can be used using thefollowing default parameters: genetic code=standard; filter=none;strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swissprotein+Spupdate+PIR. Details of these programs can be found at thefollowing internet address: http://www.ncbi.nlm.gov/cgi-bin/BLAST.

[0052] Alternatively, homology can be determined by hybridization ofpolynucleotides under conditions which form stable duplexes betweenhomologous regions, followed by digestion with single-stranded-specificnuclease(s), and size determination of the digested fragments. Two DNA,or two polypeptide sequences are “substantially homologous” to eachother when the sequences exhibit at least about 80%-85%, preferably atleast about 90%, and most preferably at least about 95%-98% sequenceidentity over a defined length of the molecules, as determined using themethods above. As used herein, substantially homologous also refers tosequences showing complete identity to the specified DNA or polypeptidesequence. DNA sequences that are substantially homologous can beidentified in a Southern hybridization experiment under, for example,stringent conditions, as defined for that particular system. Forexample, stringent hybridization conditions can include 50% formamide,5× Denhardt's Solution, 5×SSC, 0.1% SDS and 100 μg/ml denatured salmonsperm DNA and the washing conditions can include 2×SSC, 0.1% SDS at 37°C. followed by 1×SSC, 0.1% SDS at 68° C. Defining appropriatehybridization conditions is within the skill of the art. See, e.g.,Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization,supra.

[0053] The term “transdermal” delivery intends intradermal (e.g., intothe dermis or epidermis), transdermal (e.g., “percutaneous”) andtransmucosal administration, i.e., delivery by passage of an agent intoor through skin or mucosal tissue. See, e.g., Transdermal Drug Delivery:Developmental Issues and Research Initiatives, Hadgraft and Guy (eds.),Marcel Dekker, Inc., (1989); Controlled Drug Delivery: Fundamentals andApplications, Robinson and Lee (eds.), Marcel Dekker Inc., (1987); andTransdermal Delivery of Drugs, Vols. 1-3, Kydonieus and Berner (eds.),CRC Press, (1987). Thus, the term encompasses delivery of an agent usinga particle delivery device (e.g., a needleless syringe) such as thosedescribed in U.S. Pat. No. 5,630,796, as well as delivery usingparticle-mediated delivery devices such as those described in U.S. Pat.No. 5,865,796.

[0054] By “core carrier” is meant a carrier on which a guest nucleicacid (e.g., DNA, RNA) is coated in order to impart a defined particlesize as well as a sufficiently high density to achieve the momentumrequired for cell membrane penetration, such that the guest molecule canbe delivered using particle-mediated techniques (see, e.g., U.S. Pat.No. 5,100,792). Core carriers typically include materials such astungsten, gold, platinum, ferrite, polystyrene and latex. See e.g.,Particle Bombardment Technology for Gene Transfer, (1994) Yang, N. ed.,Oxford University Press, New York, N.Y. pages 10-11.

[0055] By “particle delivery device” is meant an instrument whichdelivers a particulate composition transdermally without the aid of aconventional needle to pierce the skin. Particle delivery devices foruse with the present invention are discussed throughout this document.

[0056] As used herein, the term “treatment” includes any of following:the prevention of infection or reinfection; the reduction or eliminationof symptoms; and the reduction or complete elimination of a pathogen.Treatment may be effected prophylactically (prior to infection) ortherapeutically (following infection).

[0057] The terms “individual” and “subject” are used interchangeablyherein to refer to any member of the subphylum cordata, including,without limitation, humans and other primates, including non-humanprimates such as chimpanzees and other apes and monkey species; farmanimals such as cattle, sheep, pigs, goats and horses; domestic mammalssuch as dogs and cats; laboratory animals including rodents such asmice, rats and guinea pigs; birds, including domestic, wild and gamebirds such as chickens, turkeys and other gallinaceous birds, ducks,geese, and the like. The terms do not denote a particular age. Thus,both adult and newborn individuals are intended to be covered. Themethods described herein are intended for use in any of the abovevertebrate species, since the immune systems of all of these vertebratesoperate similarly.

[0058] General Overview

[0059] Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular formulationsor process parameters as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments of the invention only, and is notintended to be limiting.

[0060] The present invention provides novel compositions containingnucleic acid sequences, wherein a first sequence in the composition is acoding sequence for an A subunit obtained or derived from anADP-ribosylating bacterial toxin, and a second sequence in thecomposition is a coding sequence for a B subunit obtained or derivedfrom an ADP-ribosylating bacterial toxin. The first and second sequencesare useful in immunization methods wherein they are delivered to asubject in order to provide for an adjuvant effect (against aco-administered antigen of interest) in the immunized subject.ADP-ribosylating bacterial toxins are a family of related bacterialexotoxins and include diphtheria toxin (DT), pertussis toxin (PT),cholera toxin (CT), the E. coli heat-labile toxins (LT1 and LT2),Pseudomonas endotoxin A, Pseudomonas exotoxin S, B. cereus exoenzyme, B.sphaericus toxin, C. botulinum C2 and C3 toxins, C. limosum exoenzyme,as well as toxins from C. perfringens, C. spiriforma and C. difficile,Staphylococcus aureus EDIN, and ADP-ribosylating bacterial toxin mutantssuch as CRM₁₉₇, a non-toxic diphtheria toxin mutant (see, e.g., Bixleret al. (1989) Adv. Exp. Med. Biol. 251:175; and Constantino et al.(1992) Vaccine). Most ADP-ribosylating bacterial toxins are organized asan A:B multimer, wherein the A subunit contains theADP-ribosyltransferase activity, and the B subunit acts as the bindingmoiety. Preferred ADP-ribosylating bacterial toxins for use in thecompositions of the present invention include cholera toxin and the E.coli heat-labile toxins.

[0061] Cholera toxin (CT) and the related E. coli heat labileenterotoxins (LT) are secretion products of their respective enterotoxicbacterial strains that are potent immunogens and exhibit strong toxicitywhen administered systemically, orally, or mucosally. Both CT and LT areknown to provide adjuvant effects for antigen when administered via theintramuscular or oral routes. These adjuvant effects have been observedat doses below that required for toxicity. The two toxins are extremelysimilar molecules, and are at least about 70-80% homologous at the aminoacid level.

[0062] The CT and LT toxins are hexamers, composed of a single moleculeof an A subunit surrounded by a doughnut-shaped ring composed of 5molecules of the B subunit. The A subunit possesses an ADP-ribosylaseactivity resulting in G protein modifications that lead to cAMP andprotein kinase A upregulation following internalization of the A subunitin a mammalian cell. The A subunit can be nicked by exogenous proteasesyielding the A1 and A2 fragments linked via a single disulfide bridge.The A subunit of CT contains a C-terminal KDEL peptide motif (RDEL forthe A subunit of LT) that is associated with retrieval of proteins fromthe trans-Golgi network into the ER. This is likely important fordelivery of the A fragment to the correct cellular compartment fortoxicity following internalization, and retention of the toxin withinthat cellular compartment. The A1 subunit enters the cell cytoplasm andtriggers Cl-efflux by catalysing ADP-ribosylation of a G protein, whichactivates adenylate cyclase leading to elevated levels of cAMP. ElevatedcAMP causes protein kinase A to phosphorylate and open the cysticfibrosis transmembrane conductance regulator chloride channel.

[0063] The A2 fragment's main role is in interacting with the B subunit.Toxin internalization is mediated by the five copies of the B subunitwhich possess binding activity for the GM1 ganglioside, aglycosphingolipid found ubiquitously on the surface of mammalian cells.

[0064] The relative importance of the A and B subunits for adjuvanticityis controversial. There is speculation in the field that the toxicactivity of the A subunit can modulate the adjuvant effects associatedwith the B subunit. Some studies demonstrate that mutations in the Asubunit abrogate adjuvant activity while others show that A subunitmutations that block ADP-ribosylase activity have no effect onadjuvanticity. Other reports show varying levels of adjuvant effects forpurified B subunits or recombinant B subunit preparations. It is likelythat the A and B subunits have separate functions that independentlycontribute to adjuvanticity. These functions are ADP-ribosylase activityand receptor triggering activity, respectively. Regarding the B subunitof LT in particular, binding to the GM1 receptor on B cells results inpolyclonal activation, occurs in the absence of significantproliferation, and involves the upregulation of a number of importantmolecules such as MHC class II, B7, CD40, ICAM-1 and IL2-Rα. For Tcells, addition of CT or LT holotoxins or recombinant B subunits toconcanavilin A-stimulated T cells results in a reduction in thymidineincorporation. Affects of these molecules on macrophages and dendriticcells have not been reported. In PBMC cultures, EtxB stimulates highlevel TNF-α and IL-10 production but not IL-12. This is consistent withthe observations of predominately Th2-like responses associated with theuse of CT and LT forms as adjuvants.

[0065] It is therefore a surprising feature of the present inventionthat administration of the present polynucleotide adjuvant compositionspreferentially produces an augmented Th1-like immune response againstthe co-administered antigen of interest, rather than the Th2-likeresponse that would be expected from the use of an ADP-ribosylatingexotoxin adjuvant composition.

[0066] Coding Sequences for ADP-Ribosylating Exotoxin Subunits

[0067] In one embodiment, a composition is provided which includes oneor more recombinant nucleic acid molecules, said one or more moleculescontaining first and second nucleic acid sequences wherein (a) the firstnucleic acid sequence is a coding region for a truncated A subunitpeptide obtained or derived from an ADP-ribosylating exotoxin, and (b)the second nucleic acid sequence is a coding region for a truncated Bsubunit peptide obtained or derived from an ADP- ribosylating exotoxin.The coding regions provide for “truncated” subunit peptides in that eachsaid subunit coding region has been genetically altered so as to createa 5′ deletion, whereby the encoded subunit peptide does not have anamino terminal bacterial signal peptide.

[0068] In another embodiment, a composition is provided which includesone or more recombinant nucleic acid molecules, said one or moremolecules containing first and second nucleic acid sequences wherein (a)the first nucleic acid sequence is a coding region for a modified,mature A subunit peptide obtained or derived from an ADP-ribosylatingexotoxin, and (b) the second nucleic acid sequence is a coding regionfor a mature B subunit peptide obtained or derived from anADP-ribosylating exotoxin. The first nucleic acid sequence contains acoding region that provides for a “modified” A subunit peptide in thatthe said coding region has been genetically altered so as to delete afour amino acid residue C-terminal KDEL or RDEL motif normally found inthe encoded subunit peptide.

[0069] In still a further embodiment, a composition is provided whichincludes one or more recombinant nucleic acid molecules, said one ormore molecules containing first and second nucleic acid sequenceswherein (a) the first nucleic acid sequence is a coding region for atruncated and modified A subunit peptide obtained or derived from anADP-ribosylating exotoxin, and (b) the second nucleic acid sequence is acoding region for a truncated B subunit peptide obtained or derived froman ADP-ribosylating exotoxin.

[0070] In particularly preferred embodiments, the ADP-ribosylatingexotoxin peptide subunit coding sequences are obtained or derived from acholera toxin (CT). In other particularly preferred embodiments, theADP-ribosylating exotoxin subunit peptide coding sequences are obtainedor derived from an E. coli heat labile enterotoxin (LT), for example LT1or LT2.

[0071] Portions of the genomes of various enterotoxic bacterial species,particularly those portions containing the coding sequences forADP-ribosylating exotoxins, are generally known and the sequencestherefor are publically available, for example on the World Wide Web,and/or are deposited with repositories such as the GenBank database. Forexample, a GenBank entry for the complete sequences of the CT subunit Aand B genes can be found at Locus VIBCTXABB (Accession No. D30053),while a GenBank entry for the complete sequences of the LT subunit A andB genes can be found at Locus AB0116677 (Accession No. AB011677). Activevariants or fragments of these toxin sequences may also be used in thecompositions and methods of the present invention. For a generaldiscussion of ADP-ribosylating exotoxins, see e.g., Krueger et al.(1995) Clin. Microbiol. Rev. 8:34-47. Furthermore, in certain aspects ofthe invention, one or both of the toxin subunit peptide coding regionscan be further genetically modified to detoxify the subunit peptide(s)encoded thereby. For example, genetically altered toxin mutants whichhave disrupted ADP-ribosylating activity, trypsin cleavage sitemutations, or disrupted binding activity are known in the art. See,e.g., Burnette et al. (1994) “Recombinant microbial ADP-ribosylatingtoxins of Bordetella pertusis, Vibrio cholerae, and enterotoxigenicEscherichia coli: structure, function and toxoid vaccine development,”in Bioprocess Technology, Burnette et al. eds., pp. 185-203; Rappuoli etal. (1995) Int. Archiv. Allergy Immunol. 108:327-333; and Rappuoli etal. (1996) Ad. Exp. Med. Biol. 397:55-60. Sequences encoding theselected toxin subunits are typically inserted into an appropriatevector (e.g., a plasmid backbone) using known techniques and asdescribed below in the Examples.

[0072] The sequence or sequences encoding the ADP-ribosylating exotoxinsubunit peptides of interest can be obtained and/or prepared using knownmethods. For example, substantially pure preparations can be obtainedusing standard molecular biological tools. That is, polynucleotidesequences coding for the above-described toxin subunuits can be obtainedusing recombinant methods, such as by screening cDNA libraries fromcells expressing the toxin subunits, or by deriving the coding sequencefor the toxin subunits from a vector known to include the same. Thetoxin subunit coding sequences from various bacterial strains are ondeposit with the American Type Culture Collection ATCC, and yet othersare available from national and international health organizations suchas the Centers of Disease Control (Atlanta, Ga.). See, e.g., Sambrook etal., supra, for a description of techniques used to obtain and isolatenucleic acid molecules. Polynucleotide sequences can also be producedsynthetically, rather than cloned.

[0073] Yet another convenient method for isolating specific nucleic acidmolecules is by the polymerase chain reaction (PCR). Mullis et al.(1987) Methods Enzymol. 155:335-350. This technique uses DNA polymerase,usually a thermostable DNA polymerase, to replicate a desired region ofDNA. The region of DNA to be replicated is identified byoligonucleotides of specified sequence complementary to opposite endsand opposite strands of the desired DNA to prime the replicationreaction. The product of the first round of replication is itself atemplate for subsequent replication, thus repeated successive cycles ofreplication result in geometric amplification of the DNA fragmentdelimited by the primer pair used.

[0074] Once the relevant sequences for the ADP-ribosylating exotoxinsubunits of interest have been obtained, they can be linked together toprovide one or more contiguous nucleic acid molecules using standardcloning or molecular biology techniques. More particularly, aftersequence information for the selected toxin subunit combination has beenobtained, the coding sequences can be combined with each other or withother sequences to form a hybrid sequence, or handled separately. Inhybrid sequences, the subunit peptide coding sequences can be positionedin any manner relative to each other, and be included in a singlemolecule in any number ways, for example, as a single copy, randomlyrepeated in the molecule as multiple copies, or included in the moleculeas multiple tandem repeats or otherwise ordered repeat motifs.

[0075] Although any number of routine molecular biology techniques canbe used to construct such recombinant nucleic acid molecules, oneconvenient method entails using one or more unique restriction sites ina shuttle or cloning vector (or inserting one or more unique restrictionsites into a suitable vector sequence) and standard cloning techniquesto direct the subunit peptide coding sequence or sequences intoparticular target locations within a vector.

[0076] Alternatively, hybrid molecules can be produced syntheticallyrather than cloned. The nucleotide sequence can be designed with theappropriate codons for the particular amino acid sequence desired. Ingeneral, one will select preferred codons for the intended host in whichthe sequence will be expressed. The complete sequence can then beassembled from overlapping oligonucleotides prepared by standard methodsand assembled into a complete coding sequence. See, e.g., Edge (1981)Nature 292:756; Nambair et al. (1984) Science (1984) 223:1299; Jay etal. (1984) J. Biol. Chem. 259:6311.

[0077] Once the relevant ADP-ribosylating exotoxin peptide codingsequences have been obtained or constructed, they can be inserted intoone or more suitable vectors which include control sequences operablylinked to the inserted sequence or sequences, thus providing expressioncassettes that allow for expression of the toxin subunit peptides invivo in a targeted subject species.

[0078] Typical promoters for mammalian cell expression include the SV40early promoter, a CMV promoter such as the CMV immediate early promoter,the mouse mammary tumor virus LTR promoter, the adenovirus major latepromoter (Ad MLP), and other suitably efficient promoter systems.Nonviral promoters, such as a promoter derived from the murinemetallothionein gene, may also be used for mammalian expression.Inducible, repressible or otherwise controllable promoters may also beused. Typically, transcription termination and polyadenylation sequenceswill also be present, located 3′ to each translation stop codon.Preferably, a sequence for optimization of initiation of translation,located 5′ to each coding sequence, is also present. Examples oftranscription terminator/polyadenylation signals include those derivedfrom SV40, as described in Sambrook et al., supra, as well as a bovinegrowth hormone terminator sequence. Introns, containing splice donor andacceptor sites, may also be designed into the expression cassette.

[0079] In addition, enhancer elements may be included within theexpression cassettes in order to increase expression levels. Examples ofsuitable enhancers include the SV40 early gene enhancer (Dijkema et al.(1985) EMBO J. 4:761), the enhancer/promoter derived from the longterminal repeat (LTR) of the Rous Sarcoma Virus (Gorman et al. (1982)Proc. Natl. Acad. Sci. USA 79:6777), and elements derived from human ormurine CMV (Boshart et al. (1985) Cell 41:521), for example, elementsincluded in the CMV intron A sequence.

[0080] In some particular embodiments, a further ancillary sequence canbe included which provides for secretion of the attachedADP-ribosylating exotoxin subunit peptide from a mammalian cell. Suchsecretion leader sequences are known to those skilled in the art, andinclude, for example, the tissue plasminogen activator (tpa) leadersignal sequence.

[0081] After one or more suitable expression cassettes (or nucleic acidconstructs such as plasmid vectors) have been produced that contain theADP-ribosylating exotoxin subunit peptide coding sequences of interest,the expression cassettes can be provided in a suitable transfectionvector such as a DNA plasmid vector or a viral vector for subsequentadministration to a subject. In this regard, the polynucleotidecompositions of the invention can be used as standalone adjuvantcompositions, or co-administered with an antigen of interest, e.g., aspart of a multi-component vaccine composition. For example, in amulti-component vaccine composition, the present nucleic acid molecules(containing the coding sequences for the ADP-ribosylating exotoxinsubunit peptides of interest) can be combined with additional nucleicacid molecules encoding one or more antigens known to be important forproviding protection against a pathogen, for example, moleculescontaining sequences that encode influenza HA or NA antigens, ormolecules containing sequences that encode HIV antigens. Alternatively,the multi-component vaccine composition may contain, in addition to thepolynucleotides encoding the toxin subunits, antigen in the form ofconventional whole virus, split virus, polysaccahride, or purifiedsubunit vaccine preparations as described herein below.

[0082] Antigens

[0083] The compositions and methods described herein are useful inadjuvanting an immune response against a wide variety of co-administeredantigens, for example antigens obtained or derived from diseased cellsor tissues, of from human or animal pathogens. For the purposes of thepresent invention, an adjuvant is used to either enhance the immuneresponse to a specific antigen, e.g., when an adjuvant isco-administered with a vaccine composition, the immune response isgreater than the immune response elicited by an equivalent amount of thevaccine composition administered without the adjuvant, or the adjuvantis used to direct a particular type or class of immune response againsta co-administered antigen. Co-administration of an “effective amount” ofthe adjuvant compositions of the present invention will be that amountwhich enhances an immunological response to the co-administered antigensuch that, for example, lower or fewer doses of the antigen are requiredto generate an efficient immune response.

[0084] As used herein, the term “co-administered,” such as when anADP-ribosylating exotoxin subunit encoding adjuvant compositionaccording to the present invention is “co-administered” with an antigenof interest (e.g., a vaccine composition), intends either thesimultaneous or concurrent administration of the adjuvant compositionand the antigen, e.g., when the two are present in the same compositionor administered in separate compositions at nearly the same time but atdifferent sites, as well as the delivery of the adjuvant composition andantigen in separate compositions at different times, including deliveryto different sites. For example, the adjuvant composition may bedelivered prior or subsequent to delivery of the antigen at the same ora different site. The timing between adjuvant and antigen deliveries canrange from about several minutes apart, to several hours apart, toseveral days apart.

[0085] For the purposes of the instant invention, the term “pathogen” isused in a broad sense to refer to a specific causative agent of adisease or condition, and includes any agent that provides a source of amolecule that elicits an immune response. Thus, pathogens include, butare not limited to, viruses, bacteria, fungi, protozoa, parasites,cancer cells and the like. Typically, the immune response is elicited byone or more peptide or carbohydrate antigens produced by the pathogen.Methods for identifying suitable antigens, obtaining and preparing suchmolecules, and then determining suitable dosages, assaying for suitableimmunogenicity and treating with such antigens are well known in theart. See e.g., Plotkin et al. (1994) Vaccines, 2^(nd) Edition, W. B.Saunders, Philadelphia, Pa. Non-limiting examples of sources forantigens that can be used to vaccinate vertebrate subjects,particularly, humans and non-human mammals, thus include viruses,bacteria, fungi, and other pathogenic organisms.

[0086] Suitable viral antigens include, but are not limited to, thoseobtained or derived from the hepatitis family of viruses, includinghepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus(HCV), the delta hepatitis virus (HDV), hepatitis E virus (HEV) andhepatitis G virus (HGV). See, e.g., International Publication Nos. WO89/04669; WO 90/11089; and WO 90/14436. The HCV genome encodes severalviral proteins, including E1 and E2. See, e.g., Houghton et al. (1991)Hepatology 14:381-388. Genomic fragments containing sequences encodingthese proteins, as well as antigenic fragments thereof, will find use inthe present methods. Similarly, the coding sequence for the δ-antigenfrom HDV is known (see, e.g., U.S. Pat. No. 5,378,814).

[0087] In like manner, a wide variety of proteins from the herpesvirusfamily can be used as antigens in the present invention, includingproteins derived from herpes simplex virus (HSV) types 1 and 2, such asHSV-1 and HSV-2 glycoproteins gB, gD and gH; antigens from varicellazoster virus (VZV), Epstein-Barr virus (EBV) and cytomegalovirus (CMV)including CMV gB and gH; and antigens from other human herpesvirusessuch as HHV6 and HHV7. (See, e.g. Chee et al. (1990) Cytomegaloviruses(J. K. McDougall, ed., Springer-Verlag, pp. 125-169; McGeoch et al.(1988) J. Gen. Virol. 69:1531-1574; U.S. Pat. No. 5,171,568; Baer et al.(1984) Nature 310:207-211; and Davison et al. (1986) J. Gen. Virol.67:1759-1816.)

[0088] Human immunodeficiency virus (HIV) antigens, such as gp120molecules for a multitude of HIV-1 and HIV-2 isolates, including membersof the various genetic subtypes of HIV, are known and reported (see,e.g., Myers et al., Los Alamos Database, Los Alamos National Laboratory,Los Alamos, N.M. (1992); and Modrow et al. (1987) J. Virol. 61:570-578)and antigen-containing genomic fragments derived or obtained from any ofthese isolates will find use in the present invention. Furthermore,other immunogenic proteins derived or obtained from any of the variousHIV isolates will find use herein, including fragments containing one ormore of the various envelope proteins such as gp160 and gp41, gagantigens such as p24gag and p55gag, as well as proteins derived from thepol, env, tat, vif, rev, nef, vpr, vpu and LTR regions of HIV.

[0089] Antigens derived or obtained from other viruses will also finduse herein, such as without limitation, antigens from members of thefamilies Picomaviridae (e.g., polioviruses, rhinoviruses, etc.);Caliciviridae; Togaviridae (e.g., rubella virus, dengue virus, etc.);Flaviviridae; Coronaviridae; Reoviridae (e.g., rotavirus, etc.);Bimaviridae; Rhabodoviridae (e.g., rabies virus, etc.); Orthomyxoviridae(e.g., influenza virus types A, B and C, etc.); Filoviridae;Paramyxoviridae (e.g., mumps virus, measles virus, respiratory syncytialvirus, parainfluenza virus, etc.); Bunyaviridae; Arenaviridae;Retroviradae (e.g., HTLV-I; HTLV-II; HIV-1 (also known as HTLV-III, LAV,ARV, hTLR, etc.)), including but not limited to antigens from theisolates HIV_(IIIb), HIV_(SF2), HIV_(LAV), HIV_(LAI), HIV_(MN));HIV-1_(CM235), HIV-1_(US4); HIV-2, among others; simian immunodeficiencyvirus (SIV); Papillomavirus, the tick-bourne encephalitis viruses; andthe like. See, e.g. Virology, 3rd Edition (W. K. Joklik ed. 1988);Fundamental Virology, 2nd Edition (B. N. Fields and D. M. Knipe, eds.1991), for a description of these and other viruses.

[0090] In some contexts, it may be preferable that the selected viralantigens are obtained or derived from a viral pathogen that typicallyenters the body via a mucosal surface and is known to cause or isassociated with human disease, such as, but not limited to, HIV (AIDS),influenza viruses (Flu), herpes simplex viruses (genital infection, coldsores, STDs), rotaviruses (diarrhea), parainfluenza viruses (respiratoryinfections), poliovirus (poliomyelitis), respiratory syncytial virus(respiratory infections), measles and mumps viruses (measles, mumps),rubella virus (rubella), and rhinoviruses (common cold).

[0091] Genomic fragments containing bacterial and parasitic antigens canbe obtained or derived from known causative agents responsible fordiseases including, but not limited to, Diptheria, Pertussis, Tetanus,Tuberculosis, Bacterial or Fungal Pneumonia, Otitis Media, Gonnorhea,Cholera, Typhoid, Meningitis, Mononucleosis, Plague, Shigellosis orSalmonellosis, Legionaire's Disease, Lyme Disease, Leprosy, Malaria,Hookworm, Onchocerciasis, Schistosomiasis, Trypamasomialsis,Lesmaniasis, Giardia, Amoebiasis, Filariasis, Borelia, and Trichinosis.Still further antigens can be obtained or derived from unconventionalviruses such as the causative agents of kuru, Creutzfeldt-Jakob disease(CJD), scrapie, transmissible mink encephalopathy, and chronic wastingdiseases, or from proteinaceous infectious particles such as prions thatare associated with mad cow disease.

[0092] Specific pathogens can include M. tuberculosis, Chlamydia, N.gonorrhoeae, Shigella, Salmonella, Vibrio Cholera, Treponema pallidua,Pseudomonas, Bordetella pertussis, Brucella, Franciscella tulorensis,Helicobacter pylori, Leptospria interrogaus, Legionella pneumophila,Yersinia pestis, Streptococcus (types A and B), Pneumococcus,Meningococcus, Hemophilus influenza (type b), Toxoplasma gondic,Complylobacteriosis, Moraxella catarrhalis, Donovanosis, andActinomycosis; fungal pathogens including Candidiasis and Aspergillosis;parasitic pathogens including Taenia, Flukes, Roundworms, Amebiasis,Giardiasis, Cryptosporidium, Schistosoma, Pneumocystis carinii,Trichomoniasis and Trichinosis. Thus, the present invention can also beused to provide a suitable immune response against numerous veterinarydiseases, such as Foot and Mouth diseases, Coronavirus, Pasteurellamultocida, Helicobacter, Strongylus vulgaris, Actinobacilluspleuropneumonia, Bovine viral diarrhea virus (BVDV), Klebsiellapneumoniae, E. coli, Bordetella pertussis, Bordetella parapertussis andbrochiseptica.

[0093] In some embodiments, the antigen of interest can be an allergen.An “allergen” is an antigen which can initiate a state ofhypersensitivity, or which can provoke an immediate hypersensitivityreaction in an individual already sensitized with the allergen.Allergens are commonly proteins or chemicals bound to proteins whichhave the property of being allergenic; however, allergens can alsoinclude organic or inorganic materials derived from a variety ofman-made or natural sources such as plant materials, metals, ingredientsin cosmetics or detergents, latexes, or the like. Classes of suitableallergens for use in the methods of the invention can include, but arenot limited to, pollens, animal dander, grasses, molds, dusts,antibiotics, stinging insect venoms, and a variety of environmental(including chemicals and metals), drug and food allergens. Common treeallergens include pollens from cottonwood, popular, ash, birch, maple,oak, elm, hickory, and pecan trees; common plant allergens include thosefrom rye, ragweed, English plantain, sorrel-dock and pigweed; plantcontact allergens include those from poison oak, poison ivy and nettles;common grass allergens include Timothy, Johnson, Bermuda, fescue andbluegrass allergens; common allergens can also be obtained from molds orfungi such as Alternaria, Fusarium, Hormodendrum, Aspergillus,Micropolyspora, Mucor and thermophilic actinomycetes; penicillin andtetracycline are common antibiotic allergens; epidermal allergens can beobtained from house or organic dusts (typically fungal in origin), frominsects such as house mites (dermatphagoides pterosinyssis), or fromanimal sources such as feathers, and cat and dog dander; common foodallergens include milk and cheese (diary), egg, wheat, nut (e.g.,peanut), seafood (e.g., shellfish), pea, bean and gluten allergens;common environmental allergens include metals (nickel and gold),chemicals (formaldehyde, trinitrophenol and turpentine), Latex, rubber,fiber (cotton or wool), burlap, hair dye, cosmetic, detergent andperfume allergens; common drug allergens include local anesthetic andsalicylate allergens; antibiotic allergens include penicillin andsulfonamide allergens; and common insect allergens include bee, wasp andant venom, and cockroach calyx allergens. Particularly wellcharacterized allergens include, but are not limited to, the major andcryptic epitopes of the Der pI allergen (Hoyne et al. (1994) Immunology83190-195), bee venom phospholipase A2 (PLA) (Akdis et al. (1996) J.Clin. Invest. 98:1676-1683), birch pollen allergen Bet v 1 (Bauer et al.(1997) Clin. Exp. Immunol. 107:536-541), and the multi-epitopicrecombinant grass allergen rKBG8.3 (Cao et al. (1997) Immunology90:46-51). These and other suitable allergens are commercially availableand/or can be readily prepared as extracts following known techniques.

[0094] Incertain other embodiments, the antigen of interest can be atumor-specific antigen. For the purposes of the present invention,tumor-specific antigens include, but are not limited to, any of thevarious MAGEs (melanoma associated antigen E), including MAGE 1, MAGE 2,MAGE 3 (HLA-A1 peptide), MAGE 4, etc.; any of the various tyrosinases(HLA-A2 peptide); mutant ras; mutant p53; and p97 melanoma antigen.Other tumor-specific antigens include the Ras peptide and p53 peptideassociated with advanced cancers, the HPV 16/18 and E6/E7 antigensassociated with cervical cancers, MUC1-KLH antigen associated withbreast carcinoma, CEA (carcinoembryonic antigen) associated withcolorectal cancer, gp100 or MART1 antigens associated with melanoma, andthe PSA antigen associated with prostate cancer. The p53 gene sequenceis known (see e.g., Harris et al. (1986) Mol. Cell. Biol. 6:4650-4656)and is deposited with GenBank under Accession No. M14694. Thus, theadjuvant compositions of the present invention can be used to carry outimmunotherapeutic methods for treating cervical, breast, colorectal,prostate, lung cancers, and melanomas.

[0095] Antigens for use with the present invention can be obtained orproduced using a variety of methods known to those of skill in the art.In particular, the antigens can be isolated directly from nativesources, using standard purification techniques. Alternatively, theantigens can be produced recombinantly using known techniques. See,e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A LaboratoryManual, Vols. I, II and II, Second Edition (1989); DNA Cloning, Vols. Iand II (D. N. Glover ed. 1985). Antigens for use herein may also besynthesized, based on described amino acid sequences, via chemicalpolymer syntheses such as solid phase peptide synthesis. Such methodsare known to those of skill in the art. See, e.g., J. M. Stewart and J.D. Young, Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co.,Rockford, Ill. (1984) and G. Barany and R. B. Merrifield, The Peptides:Analysis, Synthesis, Biology, editors E. Gross and J. Meienhofer, Vol.2, Academic Press, New York, (1980), pp. 3-254, for solid phase peptidesynthesis techniques; and M. Bodansky, Principles of Peptide Synthesis,Springer-Verlag, Berlin (1984) and E. Gross and J. Meienhofer, Eds., ThePeptides: Analysis, Synthesis, Biology, supra, Vol. 1, for classicalsolution synthesis.

[0096] If desired, polynucleotide sequences coding for theabove-described antigens, can be obtained using recombinant methods,such as by screening cDNA and genomic libraries from cells expressingthe gene, or by deriving the gene from a vector known to include thesame. Furthermore, the desired gene can be isolated directly from cellsand tissues containing the same, using standard techniques, such asphenol extraction and PCR of cDNA or genomic DNA. See, e.g., Sambrook etal., supra, for a description of techniques used to obtain and isolateDNA. Polynucleotide sequences can also be produced synthetically, ratherthan cloned.

[0097] In those compositions wherein the antigen component will beprovided by way of a nucleic acid sequence encoding an antigen ofinterest, the coding sequence for the selected antigen can be combinedwith one or both of the coding sequences for the ADP-ribosylatingexotoxin subunit peptides to provide a single construct carrying allthree coding sequences, combined with just one of the coding sequencesfor a toxin subunit to provide, e.g., a two plasmid composition, orprovided in a separate construct from either a single construct, ormultiple constructs carrying the toxin subunit coding sequences. In eachof the above cases, the antigen can be operably linked to and under thetranscriptional control of the same or different control elements, e.g.,promoters and enhancers. In those constructs where a single controlelement is used to direct transcription of two or more coding sequences,an internal ribosome entry sequence (IRES) can be used to facilitatetranscription of the multiple sequences.

[0098] Administration of Polynucleotides

[0099] The polynucleotides (nucleic acid molecules containing codingsequences for the selected ADP-ribosylating exotoxin subunit peptides)described herein may be administered by any suitable method. In apreferred embodiment, described below, the polynucleotides areadministered by coating one or more suitable constructs (e.g., one ormore DNA plasmid constructs) containing the coding sequences for theADP-ribosylating exotoxin subunit peptides of interest (and, in certainembodiments, a coding sequence for an antigen of interest on the same ora different construct) onto core carrier particles and thenadministering the coated particles to the subject or cells. However, thepolynucleotides of the present invention may also be delivered using aviral vector or using non-viral systems, e.g., naked nucleic aciddelivery.

[0100] Viral Vectors

[0101] A number of viral based systems have been used for gene delivery.For example, retroviral systems are known and generally employ packaginglines which have an integrated defective provirus (the “helper”) thatexpresses all of the genes of the virus but cannot package its owngenome due to a deletion of the packaging signal, known as the psisequence. Thus, the cell line produces empty viral shells. Producerlines can be derived from the packaging lines which, in addition to thehelper, contain a viral vector which includes sequences required in cisfor replication and packaging of the virus, known as the long terminalrepeats (LTRs). The polynucleotide molecule(s) of interest can beinserted into the vector and packaged in the viral shells synthesized bythe retroviral helper. The recombinant virus can then be isolated anddelivered to a subject. (See, e.g., U.S. Pat. No. 5,219,740.)Representative retroviral vectors include but are not limited to vectorssuch as the LHL, N2, LNSAL, LSHL and LHL2 vectors described in e.g.,U.S. Pat. No. 5,219,740, incorporated herein by reference in itsentirety, as well as derivatives of these vectors, such as the modifiedN2 vector described herein. Retroviral vectors can be constructed usingtechniques well known in the art. See, e.g., U.S. Pat. No 5,219,740;Mann et al. (1983) Cell 33:153-159.

[0102] Adenovirus based systems have been developed for gene deliveryand are suitable for delivering the nucleic acid molecules describedherein. Human adenoviruses are double-stranded DNA viruses which entercells by receptor-mediated endocytosis. These viruses are particularlywell suited for genetic transfer because they are easy to grow andmanipulate and they exhibit a broad host range in vivo and in vitro. Forexample, adenoviruses can infect human cells of hematopoietic, lymphoidand myeloid origin. Furthermore, adenoviruses infect quiescent as wellas replicating target cells. Unlike retroviruses which integrate intothe host genome, adenoviruses persist extrachromosomally thus minimizingthe risks associated with insertional mutagenesis. The virus is easilyproduced at high titers and is stable so that it can be purified andstored. Even in the replication-competent form, adenoviruses cause onlylow level morbidity and are not associated with human malignancies.Accordingly, adenovirus vectors have been developed which make use ofthese advantages. For a description of adenovirus vectors and their usessee, e.g., Haj-Ahmad and Graham (1986) J. Virol. 57:267-274; Bett et al.(1993) J. Virol. 67:5911-5921; Mittereder et al. (1994) Human GeneTherapy 5:717-729; Seth et al. (1994) J. Virol. 68:933-940; Barr et al.(1994) Gene Therapy 1:51-58; Berkner, K. L. (1988) BioTechniques6:616-629;Rich et al. (1993) Human Gene Therapy 4:461-476.

[0103] Adeno-associated viral vectors (AAV) can also be used toadminister the polynucleotide molecules described herein. In thisregard, AAV vectors can be derived from any AAV serotype, includingwithout limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAVX7, etc. AAVvectors can have one or more of the AAV wild-type genes deleted in wholeor in part, preferably the rep and/or cap genes, but retain one or morefunctional flanking inverted terminal repeat (ITR) sequences. Afunctional ITR sequence is generally deemed necessary for the rescue,replication and packaging of the AAV virion. Thus, an AAV vectorincludes at least those sequences required in cis for replication andpackaging (e.g., a functional ITR) of the virus. The ITR need not be thewild-type nucleotide sequence, and may be altered, e.g., by theinsertion, deletion or substitution of nucleotides, so long as thesequence provides for functional rescue, replication and packaging.

[0104] AAV expression vectors are constructed using known techniques toat least provide as operatively linked components in the direction oftranscription, control elements including a transcriptional initiationregion, the DNA of interest and a transcriptional termination region.The control elements are selected to be functional in a mammalian cell.The resulting construct which contains the operatively linked componentsis bounded (5′ and 3′) with functional AAV ITR sequences. Suitable AAVconstructs can be designed using techniques well known in the art. See,e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International PublicationNos. WO 92/01070 (published Jan. 23, 1992) and WO 93/03769 (publishedMar. 4, 1993); Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996;Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press);Carter, B. J. (1992) Current Opinion in Biotechnology 3:533-539;Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol.158:97-129; Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Shellingand Smith (1994) Gene Therapy 1:165-169; and Zhou et al. (1994) J. Exp.Med. 179:1867-1875.

[0105] Conventional Pharmaceutical Preparations

[0106] Formulation of a preparation comprising the polynucleotidemolecules of the present invention, with or without addition of anantigen composition, can be carried out using standard pharmaceuticalformulation chemistries and methodologies all of which are readilyavailable to the ordinarily skilled artisan. For example, compositionscontaining one or more suitable vectors can be combined with one or morepharmaceutically acceptable excipients or vehicles to provide a liquidpreparation.

[0107] Auxiliary substances, such as wetting or emulsifying agents, pHbuffering substances and the like, may be present in the excipient orvehicle. These excipients, vehicles and auxiliary substances aregenerally pharmaceutical agents that do not induce an immune response inthe individual receiving the composition, and which may be administeredwithout undue toxicity. Pharmaceutically acceptable excipients include,but are not limited to, liquids such as water, saline,polyethyleneglycol, hyaluronic acid, glycerol and ethanol.Pharmaceutically acceptable salts can also be included therein, forexample, mineral acid salts such as hydrochlorides, hydrobromides,phosphates, sulfates, and the like; and the salts of organic acids suchas acetates, propionates, malonates, benzoates, and the like. It is alsopreferred, although not required, that the preparation will contain apharmaceutically acceptable excipient that serves as a stabilizer,particularly for peptide, protein or other like antigen molecules ifthey are to be included in the vaccine composition. Examples of suitablecarriers that also act as stabilizers for peptides include, withoutlimitation, pharmaceutical grades of dextrose, sucrose, lactose,trehalose, mannitol, sorbitol, inositol, dextran, and the like. Othersuitable carriers include, again without limitation, starch, cellulose,sodium or calcium phosphates, citric acid, tartaric acid, glycine, highmolecular weight polyethylene glycols (PEGs), and combination thereof. Athorough discussion of pharmaceutically acceptable excipients, vehiclesand auxiliary substances is available in REMINGTON'S PHARMACEUTICALSCIENCES (Mack Pub. Co., N.J. 1991), incorporated herein by reference.

[0108] Certain facilitators of nucleic acid uptake and/or expression(“transfection facilitating agents”) can also be included in thecompositions, for example, facilitators such as bupivacaine, cardiotoxinand sucrose, and transfection facilitating vehicles such as liposomal orlipid preparations that are routinely used to deliver nucleic acidmolecules. Anionic and neutral liposomes are widely available and wellknown for delivering nucleic acid molecules (see, e.g., Liposomes: APractical Approach, (1990) RPC New Ed., IRL Press). Cationic lipidpreparations are also well known vehicles for use in delivery of nucleicacid molecules. Suitable lipid preparations include DOTMA(N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride),available under the tradename Lipofectin™, and DOTAP(1,2-bis(oleyloxy)-3-(trimethylammonio)propane), see, e.g., Felgner etal. (1987) Proc. Natl. Acad. Sci. USA 84:7413-7416; Malone et al. (1989)Proc. Natl. Acad. Sci. USA 86:6077-6081; U.S. Pat. Nos. 5,283,185 and5,527,928, and International Publication Nos WO 90/11092, WO 91/15501and WO 95/26356. These cationic lipids may preferably be used inassociation with a neutral lipid, for example DOPE (dioleylphosphatidylethanolamine). Still further transfection-facilitatingcompositions that can be added to the above lipid or liposomepreparations include spermine derivatives (see, e.g., InternationalPublication No. WO 93/18759) and membrane-permeabilizing compounds suchas GALA, Gramicidine S and cationic bile salts (see, e.g., InternationalPublication No. WO 93/19768).

[0109] Alternatively, the nucleic acid molecules of the presentinvention may be encapsulated, adsorbed to, or associated with,particulate carriers. Suitable particulate carriers include thosederived from polymethyl methacrylate polymers, as well as PLGmicroparticles derived from poly(lactides) andpoly(lactide-co-glycolides). See, e.g., Jeffery et al. (1993) Pharm.Res. 10:362-368. Other particulate systems and polymers can also beused, for example, polymers such as polylysine, polyarginine,polyomithine, spermine, spermidine, as well as conjugates of thesemolecules.

[0110] The formulated compositions will thus typically include one ormore polynucleotide molecule (e.g., plasmid vector) containing thecoding sequences for the selected ADP-ribosylating exotoxin subunitpeptides in an amount sufficient to adjuvant an immunological responseagainst a co-administered antigen. An appropriate effective amount canbe readily determined by one of skill in the art. Such an amount willfall in a relatively broad range that can be determined through routinetrials. For example, suitable adjuvant effect may be obtained using aslittle as 0. 1 μg of DNA, while in other administrations, up to 2 mg ofDNA may be used. It is generally expected that an effective dose ofpolynucleotides containing the ADP-ribosylating exotoxin subunit peptidecoding sequences of interest will fall within a range of about 1 μg to1000 μg, however, doses above and below this range may also be foundeffective. The compositions may thus contain from about 0.1% to about99.9% of the polynucleotide molecules.

[0111] Administration of Conventional Pharmaceutical Preparations

[0112] Administration of the above-described pharmaceutical preparationscan be effected in one dose, continuously or intermittently throughoutthe course of treatment. That is, once suitably formulated, thecompositions of the present invention can be administered to a subjectin vivo using a variety of known routes and techniques. For example, theliquid preparations can be provided as an injectable solution,suspension or emulsion and administered via parenteral, subcutaneous,intradermal, intramuscular, intravenous injection using a conventionalneedle and syringe, or using a liquid jet injection system. Liquidpreparations can also be administered topically to skin or mucosaltissue, or provided as a finely divided spray suitable for respiratoryor pulmonary administration. Other modes of administration include oraladministration, suppositories, and active or passive transdermaldelivery techniques.

[0113] Alternatively, the compositions can be administered ex vivo, forexample delivery and reimplantation of transformed cells into a subjectare known (e.g., dextran-mediated transfection, calcium phosphateprecipitation, electroporation, and direct microinjection of intonuclei). However, delivery will most typically be via conventionalneedle and syringe for the liquid compositions and for liquidsuspensions containing particulate compositions. Methods of determiningthe most effective means and dosages of administration are well known tothose of skill in the art and will vary with the delivery vehicle, thecomposition of the therapy, the target cells, and the subject beingtreated. Single and multiple administrations can be carried out with thedose level and pattern being selected by the attending physician. Itshould be understood that more than one subunit coding region and/orantigen coding region can be carried by a single polynucleotide vectorconstruct. Alternatively, separate vectors (e.g., viral vectors,plasmids, or any combination thereof), each expressing one or more toxinsubunit peptide and/or antigen derived from any pathogen can also bedelivered to a subject as described herein.

[0114] Furthermore, it is also intended that the polynucleotidesdelivered by the methods of the present invention be combined with othersuitable compositions and therapies. For instance, in order to furtheraugment an immune response in a subject, the compositions and methodsdescribed herein can be combined with delivery of ancillary substances(e.g., other adjuvants), such as pharmacological agents, cytokines, orthe like. Ancillary substances may be administered, for example, asproteins or other macromolecules at the same time, prior to, orsubsequent to, administration of the polynucleotiode molecules describedherein. The nucleic acid molecule compositions may also be administereddirectly to the subject or, alternatively, delivered ex vivo, to cellsderived from the subject, using methods known to those skilled in theart.

[0115] Coated Particles

[0116] In one embodiment, polynucleotide constructs containing thecoding sequences for the selected ADP-ribosylating exotoxin subunitpeptides, and other ancillary components (such as coding sequences forone or more antigens) are delivered using carrier particles.Particle-mediated delivery methods for administering such nucleic acidpreparations are known in the art. Thus, once prepared and suitablypurified, the above-described plasmid vector constructs can be coatedonto carrier particles (e.g., core carriers) using a variety oftechniques known in the art. Carrier particles are selected frommaterials which have a suitable density in the range of particle sizestypically used for intracellular delivery from an appropriate particledelivery device. The optimum carrier particle size will, of course,depend upon the diameter of the target cells.

[0117] For the purposes of the present invention, tungsten, gold,platinum and iridium core carrier particles can be used. Tungsten andgold particles are preferred. Tungsten particles are readily availablein average sizes of 0.5 to 2.0 μm in diameter. Although such particleshave optimal density for use in particle delivery methods, and allowhighly efficient coating with DNA, tungsten may potentially be toxic tocertain cell types. Accordingly, gold particles or microcrystalline gold(e.g., gold powder A1570, available from Engelhard Corp., East Newark,N.J.) will also find use with the present methods. Gold particlesprovide uniformity in size (available from Alpha Chemicals in particlesizes of 1-3 μm, or available from Degussa, South Plainfield, N.J. in arange of particle sizes including 0.95 μm) and reduced toxicity.

[0118] A number of methods are known and have been described for coatingor precipitating DNA or RNA onto gold or tungsten particles. Most suchmethods generally combine a predetermined amount of gold or tungstenwith plasmid DNA, CaCl₂ and spermidine. The resulting solution isvortexed continually during the coating procedure to ensure uniformityof the reaction mixture. After precipitation of the nucleic acid, thecoated particles can be transferred to suitable membranes and allowed todry prior to use, coated onto surfaces of a sample module or cassette,or loaded into a delivery cassette for use in a suitable particledelivery device.

[0119] Peptide antigens can also be coated onto the same or similar corecarrier particles. For example, peptides can be attached to a carrierparticle by simply mixing the two components in an empiricallydetermined ratio, by ammonium sulfate precipitation or other solventprecipitation methods familiar to those skilled in the art, or bychemical coupling of the peptide to the carrier particle. The couplingof L-cysteine residues to gold has been previously described (Brown etal., Chemical Society Reviews 9:271-311 (1980)). Other methods wouldinclude, for example, dissolving the peptide in absolute ethanol, water,or an alcohol/water mixture, adding the solution to a quantity ofcarrier particles, and then drying the mixture under a stream of air ornitrogen gas while vortexing. Alternatively, the antigen can be driedonto carrier particles by centrifugation under vacuum. Once dried, thecoated particles can be resuspended in a suitable solvent (e.g., ethylacetate or acetone), and triturated (e.g., by sonication) to provide asubstantially uniform suspension. The core carrier particles coated withthe antigen can then be combined with core carrier particles carryingthe ADP-ribosylating exotoxin subunit peptide construct(s) andadministered in a single particle injection step, or administeredseparately from the toxin subunit compositions.

[0120] Administration of Coated Particles

[0121] Following their formation, core carrier particles coated with thenucleic acid preparations of the present invention, alone or incombination with e.g., antigen preparations, are delivered to a subjectusing particle-mediated delivery techniques.

[0122] Various particle delivery devices suitable for particle-mediateddelivery techniques are known in the art, and are all suited for use inthe practice of the invention. Current device designs employ anexplosive, electric or gaseous discharge to propel the coated corecarrier particles toward target cells. The coated particles canthemselves be releasably attached to a movable carrier sheet, orremovably attached to a surface along which a gas stream passes, liftingthe particles from the surface and accelerating them toward the target.An example of a gaseous discharge device is described in U.S. Pat. No.5,204,253. An explosive-type device is described in U.S. Pat. No.4,945,050. One example of an electric discharge-type particleacceleration apparatus is described in U.S. Pat. No. 5,120,657. Anotherelectric discharge apparatus suitable for use herein is described inU.S. Pat. No. 5,149,655. The disclosure of all of these patents isincorporated herein by reference in their entireties.

[0123] The coated particles are administered to the subject to betreated in a manner compatible with the dosage formulation, and in anamount that will be effective to bring about the desired adjuvanteffect/immune response. The amount of the composition to be deliveredwhich, in the case of nucleic acid molecules is generally in the rangeof from 0.001 to 1000 μg, more typically 0.01 to 10.0 μg of nucleic acidmolecule per dose, and in the case of peptide or protein molecules is 1μg to 5 mg, more typically 1 to 50 μg of peptide, depends on the subjectto be treated. The exact amount necessary will vary depending on the ageand general condition of the individual being immunized and theparticular nucleotide sequence or peptide selected, as well as otherfactors. An appropriate effective amount can be readily determined byone of skill in the art upon reading the instant specification.

[0124] Particulate Compositions

[0125] Alternatively, polynucleotides carrying the selectedADP-ribosylating exotoxin subunit peptide coding sequences, as well asone or more selected antigen moiety, can be formulated as a particulatecomposition. More particularly, formulation of particles comprising oneor more polynucleotide molecules can be carried out using standardpharmaceutical formulation chemistries and methodologies all of whichare readily available to the reasonably skilled artisan. For example,one or more vector construct and/or antigen component can be combinedwith one or more pharmaceutically acceptable excipients or vehicles toprovide a vaccine composition. Auxiliary substances, such as wetting oremulsifying agents, pH buffering substances, and the like, may bepresent in the excipient or vehicle. These excipients, vehicles andauxiliary substances are generally pharmaceutical agents that do notthemselves induce an immune response in the individual receiving thecomposition, and which may be administered without undue toxicity.Pharmaceutically acceptable excipients include, but are not limited to,liquids such as water, saline, polyethyleneglycol, hyaluronic acid,glycerol and ethanol. Pharmaceutically acceptable salts can be includedtherein, for example, mineral acid salts such as hydrochlorides,hydrobromides, phosphates, sulfates, and the like; and the salts oforganic acids such as acetates, propionates, malonates, benzoates, andthe like. It is also preferred, although not required, that the nucleicacid composition will contain a pharmaceutically acceptable carrier thatserves as a stabilizer, particularly for peptide, protein or other likeantigens or ancillary materials. Examples of suitable carriers that alsoact as stabilizers for peptides include, without limitation,pharmaceutical grades of dextrose, sucrose, lactose, trehalose,mannitol, sorbitol, inositol, dextran, and the like. Other suitablecarriers include, again without limitation, starch, cellulose, sodium orcalcium phosphates, citric acid, tartaric acid, glycine, high molecularweight polyethylene glycols (PEGs), and combination thereof. A thoroughdiscussion of pharmaceutically acceptable excipients, carriers,stabilizers and other auxiliary substances is available in REMINGTON'SPHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991), incorporated hereinby reference.

[0126] The formulated compositions will include an amount of the toxinsubunit peptide-encoding polynucleotides which is sufficient to providethe desired adjuvant effect against a co-administered antigen, asdefined above. An appropriate effective amount can be readily determinedby one of skill in the art. Such an amount will fall in a relativelybroad range, generally within the range of about 0.001 μg to 25 mg ormore of the nucleic acid construct of interest, and specific suitableamounts can be determined through routine trials. The compositions maycontain from about 0.1% to about 99.9% of the nucleic acid molecule(s).If an antigen component is included in the composition, or the methodsare used to provide a particulate antigen composition, the antigen willbe present in a suitable amount as described above. The compositions arethen prepared as particles using standard techniques, such as by simpleevaporation (air drying), vacuum drying, spray drying, freeze drying(lyophilization), spray-freeze drying, spray coating, precipitation,supercritical fluid particle formation, and the like. If desired, theresultant particles can be densified using the techniques described incommonly owned International Publication No. WO 97/48485, incorporatedherein by reference.

[0127] Single unit dosages or multidose containers, in which theparticles may be packaged prior to use, can comprise a hermeticallysealed container enclosing a suitable amount of the particles comprisinga suitable nucleic acid construct and/or a selected antigen (e.g., toprovide a multicomponent vaccine composition). The particulatecompositions can be packaged as a sterile formulation, and thehermetically sealed container can thus be designed to preserve sterilityof the formulation until use in the methods of the invention. Ifdesired, the containers can be adapted for direct use in a particledelivery device. Such containers can take the form of capsules, foilpouches, sachets, cassettes, and the like. Appropriate particle deliverydevices (e.g., needleless syringes) are described herein.

[0128] The container in which the particles are packaged can further belabelled to identify the composition and provide relevant dosageinformation. In addition, the container can be labelled with a notice inthe form prescribed by a governmental agency, for example the Food andDrug Administration, wherein the notice indicates approval by the agencyunder Federal law of the manufacture, use or sale of the adjuvant,antigen (or vaccine composition) contained therein for humanadministration.

[0129] The particulate compositions can then be administered using atransdermal delivery technique. Preferably, the particulate compositionswill be delivered via a powder injection method, e.g., delivered from aneedleless syringe system such as those described in commonly ownedInternational Publication Nos. WO 94/24263, WO 96/04947, WO 96/12513,and WO 96/20022, all of which are incorporated herein by reference.Delivery of particles from such needleless syringe systems is typicallypractised with particles having an approximate size generally rangingfrom 0.1 to 250 μm, preferably ranging from about 10-70 μm. Particleslarger than about 250 μm can also be delivered from the devices, withthe upper limitation being the point at which the size of the particleswould cause untoward damage to the skin cells. The actual distance whichthe delivered particles will penetrate a target surface depends uponparticle size (e.g., the nominal particle diameter assuming a roughlyspherical particle geometry), particle density, the initial velocity atwhich the particle impacts the surface, and the density and kinematicviscosity of the targeted skin tissue. In this regard, optimal particledensities for use in needleless injection generally range between about0.1 and 25 g/cm³, preferably between about 0.9 and 1.5 g/cm³, andinjection velocities generally range between about 100 and 3,000 m/sec,or greater. With appropriate gas pressure, particles having an averagediameter of 10-70 μm can be accelerated through the nozzle at velocitiesapproaching the supersonic speeds of a driving gas flow.

[0130] Compositions containing a therapeutically effective amount of thepowdered molecules described herein can be delivered to any suitabletarget tissue via the above-described particle delivery devices. Forexample, the compositions can be delivered to muscle, skin, brain, lung,liver, spleen, bone marrow, thymus, heart, lymph, blood, bone cartilage,pancreas, kidney, gall bladder, stomach, intestine, testis, ovary,uterus, rectum, nervous system, eye, gland and connective tissues. Fornucleic acid molecules, delivery is preferably to, and the moleculesexpressed in, terminally differentiated cells; however, the moleculescan also be delivered to non-differentiated, or partially differentiatedcells such as stem cells of blood and skin fibroblasts.

[0131] If desired, these needleless syringe systems can be provided in apreloaded condition containing a suitable dosage of the particlescomprising the ADP-ribosylating exotoxin subunit peptide codingsequences of interest, The loaded syringe can be packaged in ahermetically sealed container, which may further be labelled asdescribed above.

[0132] Thus, the method can be used to obtain nucleic acid particleshaving a size ranging from about 10 to about 250 μm, preferably about 10to about 150 μm, and most preferably about 20 to about 60 μm; and aparticle density ranging from about 0.1 to about 25 g/cm³, and a bulkdensity of about 0.5 to about 3.0 g/cm³, or greater.

[0133] Similarly, particles of selected antigens having a size rangingfrom about 0.1 to about 250 μm, preferably about 0.1 to about 150 μm,and most preferably about 20 to about 60 μm; a particle density rangingfrom about 0.1 to about 25 g/cm³, and a bulk density of preferably about0.5 to about 3.0 g/cm³, and most preferably about 0.8 to about 1.5 g/cm³can be obtained.

[0134] Enhancing Immune Responses

[0135] In another embodiment of the invention, a method for enhancing animmune response against a co-administered antigen of interest isprovided. In essence, the method entails (a) administering an antigen ofinterest to a subject, (b) providing an adjuvant composition accordingto the present invention, wherein the said composition contains one ormore nucleic acid molecules containing selected coding sequences forADP-ribosylating exotoxin subunit peptides, and (c) co-administering theadjuvant composition to the subject, whereby the toxin subunit peptidesare expressed from their respective coding sequences in an amountsufficient to elicit an enhanced immune response against theco-administered antigen. As described in detail herein above, the codingsequences are operably linked to the same or different regulatorysequences to provide one or more expression cassettes. These expressioncassettes are then provided in a suitable vector, for example a plasmidvector construct or a viral vector.

[0136] In one aspect, the method entails administering thepolynucleotide composition to a subject using standard gene deliverytechniques that are known in the art. See, e.g., U.S. Pat. Nos.5,399,346, 5,580,859, 5,589,466. Typically, the polynucleotide vaccinecomposition is combined with a pharmaceutically acceptable excipient orvehicle to provide a liquid preparation (as described herein above) andthen used as an injectable solution, suspension or emulsion foradministration via parenteral, subcutaneous, intradermal, intramuscular,intravenous injection using a conventional needle and syringe, or usinga liquid jet injection system. It is preferred that the composition beadministered to skin or mucosal tissue of the subject. Liquidpreparations can also be administered topically to skin or mucosaltissue, or provided as a finely divided spray suitable for respiratoryor pulmonary administration. Other modes of administration include oraladministration, suppositories, and active or passive transdermaldelivery techniques. The polynucleotide compositions can alternativelybe delivered ex vivo to cells derived from the subject, whereafter thecells are reimplanted in the subject. Upon introduction into thesubject, the nucleic acid sequence is expressed to provide theADP-ribosylating exotoxin subunit peptides in situ in an amountsufficient to enhance an immune response against the co-administeredantigen in the vaccinated subject. This enhanced immune response can becharacterized as an enhanced humoral (antibody) response, an enhancedcellular (CTL) response, or be characterized as enhancing both humoraland cellular responses against the co-administered antigen.

[0137] In certain preferred embodiments, the enhanced immune response ischaracterized as an augmented Th1-like (cellular) immune responseagainst the co-administered antigen, rather than a Th2-like response. Inthis regard, the augmented Th1-like immune response can be qualified byone or more of the following: increased titers of interferon-γ producingCD4⁺ helper T lymphocytes and/or CD8⁺ CTLs; increased antigen-specificCTL activity; of increased titers of antigen-specific antibodies of thesubclass typically associated with cellular immunity (e.g., IgG2a).

[0138] It is preferred that the polynucleotide adjuvant compositions ofthe present invention be delivered in particulate form. For example, thecompositions can be administered using a particle delivery device asdescribed in detail herein above. In certain embodiments, thepolynucleotide compositions can be coated onto core carrier particlesusing a variety of techniques known in the art and delivered using aparticle-mediated delivery method. Carrier particles are selected frommaterials which have a suitable density in the range of particle sizestypically used for intracellular delivery from a particle deliverydevice. The optimum carrier particle size will, of course, depend on thediameter of the target cells.

[0139] These methods can alternatively be modified by co-administrationof additional or ancillary components to the subject. For example, asecondary vaccine composition can be administered, wherein the secondarycomposition can comprise a nucleic acid vaccine, or the secondaryvaccine composition can comprise a conventional vaccine such as a wholevirus, split virus, or subunit vaccine. The secondary vaccinecomposition can be combined with the polynucleotide ADP-ribosylatingexotoxin subunit peptide compositions to form a single composition, orthe secondary vaccine composition can be administered separately to thesame or to a different site, either concurrently, sequentially, orseparated by a significant passage of time such as in a boosting stepsome days after the initial composition has been administered.

[0140] As above, the secondary vaccine composition and/or otherancillary component can be administered by injection using either aconventional syringe, or using a particle-mediated delivery system asalso described above. Administration will typically be eithersubcutaneously, epidermally, intradermally, intramucosally (e.g.,nasally, rectally and/or vaginally), intraperitoneally, intravenously,orally or intramuscularly. Other modes of administration includetopical, oral and pulmonary administration, suppositories, andtransdermal applications. Dosage treatment may be a single dose scheduleor a multiple dose schedule.

EXPERIMENTAL

[0141] Below are examples of specific embodiments for carrying out thepresent invention. The examples are offered for illustrative purposesonly, and are not intended to limit the scope of the present inventionin any way.

[0142] Efforts have been made to ensure accuracy with respect to numbersused (e.g., amounts, temperatures, etc.), but some experimental errorand deviation should, of course, be allowed for.

Example 1 Development of Expression Vectors Encoding the A and BSubunits of CT

[0143] Genomic DNA from Vibrio cholerae was used as a template forpolymerase chain reactions (PCR) to generate DNA fragments containingthe coding sequences for both the A and B subunits of cholera toxin(CT). For PCR generation of the A subunit-encoding fragment (CTA), thefollowing two oligodeoxyribonucleotide primers were used:

[0144] Primer 1: 5′-GGA GCT AGC AAT GAT GAT AAG TTA TAT CGG-3′ (SEQ IDNO: 7); and

[0145] Primer 2: 5′-CCT GGA TCC TCA TAA TTC ATC CTT AAT TCT-3′ (SEQ IDNO: 8).

[0146] In order to facilitate insertion into an expression vector,Primers 1 and 2 contain extra sequences at their 5′ ends (outside theregion of homology to the CTA coding sequence) which include recognitionsites for NheI and BamHI, respectively. Primers 1 and 2 were designed tolead to PCR generation of a fragment of the A subunit coding sequencestarting at nucleotide position 164 and ending at nucleotide position886 (GenBank Accession #D30053). This region encompasses the entirecoding sequence for the mature subunit A peptide but does not includethe sequence encoding the bacterial signal peptide found at the aminoterminus of the pre-subunit A peptide.

[0147] For PCR generation of the B subunit-encoding fragment (CTB), thefollowing two oligodeoxyribonucleotide primers were used:

[0148] Primer 3: 5′-GGA GCT AGC ACA CCT CAA AAT ATT ACT GAT-3′ (SEQ IDNO: 9); and

[0149] Primer 4: 5′-CCT GGA TCC TTA ATT TGC CAT ACT AAT TGC-3′ (SEQ IDNO: 10).

[0150] In order to facilitate insertion into an expression vector,Primers 3 and 4 contain extra sequences at their 5′ ends (outside theregion of homology to the CTB coding sequence) which include recognitionsites for NheI and BamHI, respectively. Primers 3 and 4 were designed tolead to PCR generation of a fragment of the B subunit coding sequencestarting at nucleotide position 946 and ending at nucleotide position1257 (GenBank Accession #D30053). This region encompasses the entirecoding sequence for the mature subunit B peptide but does not includethe sequence encoding the bacterial signal peptide found at the aminoterminus of the pre-subunit B peptide.

[0151] In addition to the two PCR reactions described above, a third PCRreaction was performed to generate a coding sequence for a modified formof subunit A of CT in which the C-terminal four amino acid KDEL motifwas eliminated. This reaction involved the use of Primer 1 (SEQ ID NO:7) and the following primer:

[0152] Primer 5: 5′-CCT GGA TCC TCA AAT TCT ATT ATG TGT ATC-3′ (SEQ IDNO: 11).

[0153] All PCR reactions were performed using Pfu Turbo DNA polymerase(obtained from Strategene, La Jolla, Calif.) along with the PCR reactionbuffer supplied by the manufacturer. PCR conditions were as follows: 95°C. for 2 min., 30 cycles of (95° C. for 1 min., 55° C. for 2 min. 15sec., 72° C. for 1 min.), 72° C. for 5 min., and 4° C. hold.

[0154] Following completion of the PCR reactions, the newly synthesizedfragments were digested with NheI and BamHI enzymes to generate cohesiveends, and the individual fragments were inserted into the NheI- andBamHI-cleaved pWRG7054 expression vector resulting in the followingclones: pPJV2002, pPJV2003, and pPJV2006 encoding CTA, CTB, and modifiedCTA (minus KDEL), respectively. The parental cloning vector pWRG7054contains the human cytomegalovirus immediate early promoter with theassociated intron A sequence. In addition, the coding sequence for thesignal peptide of human tissue plasminogen activator is included inpWRG7054 to allow for the secretion from mammalian cells of any proteinwhose coding sequence is inserted at the NheI site in the appropriatereading frame. (See, e.g., Chapman et al. (1991) Nuc. Acids Res.19:3979-3986, and Burke et al. (1986) J. Biol. Chem. 261:12574-12578.)

[0155] The restriction maps and full sequences of plasmids pPJV2002,pPJV2003, and pPJV2006 are depicted in FIGS. 1, 2, and 3, respectively.

Example 2 Development of Expression Vectors Encoding the A and BSubunits of E. coli Heat Labile Enterotoxin

[0156] Genomic DNA from E. coli strain E078:H11 (American Type CultureCollection #35401) was used as a template for polymerase chain reactionsto generate DNA fragments containing the coding sequences for both the Aand B subunits of the heat labile enterotoxin (LT). For PCR generationof the A subunit-encoding fragment, the following twooligodeoxyribonucleotide primers were used:

[0157] Primer 6: 5′-GGA GCT AGC AAT GGC GAC AAA TTA TAC CGT-3′ (SEQ IDNO: 12); and

[0158] Primer 7: 5′-CCT GGA TCC TCA TAA TTC ATC CCG AAT TCT-3′ (SEQ IDNO: 13).

[0159] In order to facilitate insertion into an expression vector,Primers 6 and 7 contain extra sequences at their 5′ ends (outside theregion of homology to the LTA coding sequence) which include recognitionsites for NheI and BamHI, respectively. Primers 6 and 7 were designed tolead to PCR generation of a fragment of the A subunit coding sequencestarting at nucleotide position 145 and ending at nucleotide position867 of the sequence found in the GenBank database (Accession #AB011677).This region encompasses the entire coding sequence for the maturesubunit A peptide but does not include the sequence encoding thebacterial signal peptide found at the amino terminus of the pre-subunitA peptide.

[0160] For PCR generation of the B subunit-encoding fragment, thefollowing two oligodeoxyribonucleotide primers were used:

[0161] Primer 8: 5′-GGA GCT AGC GCT CCC CAG TCT ATT ACA GAA-3′ (SEQ IDNO: 14); and

[0162] Primer 9: 5′-CCT GGA TCC CTA GTT TTC CAT ACT GAT TGC-3′ (SEQ IDNO: 15).

[0163] In order to facilitate insertion into an expression vector,Primers 8 and 9 contain extra sequences at their 5′ ends (outside theregion of homology to the LTB coding sequence) which include recognitionsites for NheI and BamHI, respectively. Primers 8 and 9 were designed tolead to PCR generation of a fragment of the B subunit coding sequencestarting at nucleotide position 927 and ending at nucleotide position1238 of the sequence found in the GenBank database (Accession#AB011677). This region encompasses the entire coding sequence for themature subunit B peptide but does not include the sequence encoding thebacterial signal peptide found at the amino terminus of the pre-subunitB peptide.

[0164] In addition to the two PCR reactions described above, a third PCRreaction was performed to generate a coding sequence for a modified formof subunit A of LT in which the C-terminal four amino acid RDEL motifwas eliminated. This reaction involved the use of Primer 6 (SEQ ID NO:12) and the following primer:

[0165] Primer 10: 5′-CCT GGA TCC TCA AAT TCT GTT ATA TAT GTC-3′ (SEQ IDNO: 16).

[0166] All PCR reactions were performed using Pfu Turbo DNA polymerase(obtained from Strategene, La Jolla, Calif.) along with the PCR reactionbuffer supplied by the manufacturer. PCR conditions were as follows: 95°C. for 2 min., 30 cycles of (95° C. for 1 min., 55° C. for 2 min. 15sec., 72° C. for 1 min.), 72° C. for 5 min., 4° C. hold.

[0167] Following completion of the PCR reactions, the newly synthesizedfragments were digested with NheI and BamHI to generate cohesive endsand the individual fragments were inserted into the NheI- andBamHI-cleaved pWRG7054 expression vector resulting in clones pPJV2004,pPJV2005, and pPJV2007 encoding LTA, LTB, and modified LTA (minus RDEL),respectively.

[0168] The restriction maps and complete sequences of plasmids pPJV2004,pPJV2005, and pPJV2007 are shown in FIGS. 4, 5 and 6, respectively.

Example 3 Enhancement of Antigen-Specific Antibody Response to a DNAVaccine Using Plasmid Vectors Encoding CTA and/or CTB

[0169] A DNA vaccine vector encoding the M2 protein of influenza A viruswas employed to test the adjuvant effects of the pPJV2002, pPJV2003, andpPJV2006 adjuvant vectors in the context of particle-mediated DNAvaccination. Particle-mediated DNA vaccination was performed byprecipitating the M2 DNA vaccine vector, with our without variouscombinations of the pPJV2002, pPJV2003 and pPJV2006 adjuvant vectors,onto microscopic gold particles and accelerating the coated goldparticles into the epidermis of mice using a PowderJect® XR-1 particledelivery device (PowderJect Vaccines, Inc. Madison, Wis.).

[0170] More particularly, the sequence from the RNA segment #7 (thatencodes the M2 protein) of influenza virus strain A/Kagoshima/10/95(H3N2) was used as a model to design PCR primers to facilitate cloningof the mature M2 coding sequence from A/Sydney/5/97 (H3N2). TheA/Kagoshima sequence was used for primer design since the sequence ofRNA segment 7 of A/Sydney has not yet been determined. The high degreeof conservation among M2 sequences was expected to facilitate the use ofprimers designed from a different viral strain.

[0171] Since M2 is translated from a spliced RNA, it was deemednecessary that nucleotide positions 27 to 714 in the coding region ofsegment 7 RNA were spliced out. Accordingly, a set of PCR primers wasgenerated and designed to generate the complete M2 coding sequence butensure that the intron was cleanly eliminated from resulting M2 codingsequence clone. The PCR primers used to generate the full-length M2coding sequence clone were as follows:

[0172] Primer 11: 5′-CCC AAG CTT CCA CCA TGA GCC TTC TAA CCG AGG TCG AAACAC CTA TCA GAA ACG AAT GGG AGT GC-3′ (SEQ ID NO: 17); and

[0173] Primer 12: 5′-CCC GGA TCC TTA CTC CAG CTC TAT GCT G-3′ (SEQ IDNO: 18).

[0174] Primer 11 (SEQ ID NO: 17) contains additional sequences at its 5′end that include a recognition site for HindIII and a Kozak consensussequence to facilitate mRNA translation initiation. Also, Primer 12 (SEQID NO:) contains additional sequences at its 5′ end that includes arecognition sequence for BamHI.

[0175] Viral RNA was isolated from a sample of A/Sydney/5/97 (H3N2) thatwas grown in embryonated chicken eggs. The viral RNA isolation processused standard techniques known to those skilled in the art. RNA fromthis virus was used in a reverse transcriptase/polymerase chain reaction(RT-PCR) using an RT-PCR kit obtained from Stratagene (La Jolla,Calif.). The RT reaction step was completed by adding 5.9 μl ofRNase-free water to a reaction tube. To this tube was added 1.0 μl 10×MMLV-RT buffer and 1.0 μl dNTP mix from the kit. Also, 1 μl ofA/Sydney/5/97 RNA and 0.6 μl (0.6 μg) of Primer 11 (SEQ ID NO: 17) wasadded. The reaction was heated to 65° C. for 5 minutes to denature theRNA, after which 0.5 μl of reverse transcriptase from the kit was added.The reaction was incubated at 37° C. for 15 minutes to complete thereverse transcription step.

[0176] The PCR reaction step was completed by addition of the followingcomponents to a new reaction tube: 40 μl water; 5 μl 10× ultra HF bufferfrom the kit; 1.0 μl dNTP mix from the kit; 1.0 μl Primer 11 (SEQ ID NO:17) (1.0 μg); 1.0 μl Primer 12 (SEQ ID NO: 18) (1.0 μg); 1 μl of thereverse transcriptase reaction mix from above; and 1 μl Turbo PFUpolymerase from the kit. The PCR reaction was carried out using thefollowing incubation scheme: 1 minute @ 95° C.; followed by 30 cycles of(30 sec @ 95° C., 30 sec @ 46° C., 3 min @ 68° C.), followed by 10minutes @ 68° C. PCR products were electrophoresed on a 2% agarose gelrevealing a single DNA band of the expected size of approximately 300bp.

[0177] The approximately 300 bp band was isolated from the gel anddigested with HindIII and BamHI in order to generate the necessarysticky ends for insertion into the pWRG7077 DNA vaccine expressionvector (Schmaljohn et al. (1997) J. Virol. 71:9563-9569). The pWRG7077DNA was digested partially with HindIII and completely with BamHI tofacilitate insertion of the M2 coding insert. The requirement for apartial HindIII digestion of the vector was due to the presence of asecond HindIII site in the Kanamycin resistance marker of this plasmid.The resulting M2 DNA vaccine vector was termed pM2-FL. The pM2-FL vectorcontains the immediate early promoter from human cytomegalovirus (hCMV)and its associated intron A sequence to drive transcription from the M2coding sequence. This vector also includes a polyadenylation sequencefrom the bovine growth hormone gene.

[0178] The pM2-FL plasmid was then precipitated onto 2 micron goldparticles as single vector, or mixed vector plus adjuvant vector samples(i.e., the M2 plasmid vector was combined with pPJV2002, pPJV2003,and/or pPJV2006 adjuvant vectors). Specifically, plasmid DNA (single M2vector or M2 vector plus one or more adjuvant vectors) was mixed with 2micron gold particles (Degussa, Lot 65-0) in a small centrifuge tubecontaining 400 μl of 50 mM spermidine. The DNA-to-gold ratio varied from2.5 μg DNA per mg of gold to 4.0 μg of DNA per mg of gold, and a singlebatch contained 26 mg of gold. DNA was precipitated onto the goldparticles by addition of a 1/10 volume of 10% CaCl₂ during continuousagitation of the tube on a rotary mixer. DNA-gold complexes were washedthree times with absolute ethanol, and then injected into a TEFZEL® tube(McMaster-Carr) housed in a tube turner coating machine (PowderJectVaccines, Inc., Madison Wis.) which coats the inside of the tube withthe gold/DNA complex. This tube turner machine is described in U.S. Pat.No. 5,733,600. See also PCT patent application PCT/US95/00780 and U.S.Pat. Nos. 5,780,100; 5,865,796 and 5,584,807 After the coating procedurewas completed, the tubes were cut into 0.5 inch “cartridges” suitablefor loading into a particle delivery device.

[0179] The following DNA-gold formulations were generated for a mouseDNA vaccine adjuvant trial.

[0180] Formulation #1: pM2-FL DNA vector alone, 2.5 μg DNA per mg gold,0.5 mg gold per cartridge;

[0181] Formulation #2: pM2-FL DNA vector precipitated onto one batch ofgold (2 μg pM2-FL DNA per mg gold), pPJV2002 and pPJV2003 DNA vectorscoprecipitated onto a second batch of gold (1.75 μg of each DNA adjuvantvector per mg gold), the gold batches were mixed equally, 0.5 mg goldper cartridge;

[0182] Formulation #3: pM2-FL DNA vector, pPJV2002 and pPJV2003 DNAvectors all coprecipitated onto single batch of gold (2 μg pM2-FL DNAper mg gold, and 1 μg each of pPJV2002 and pPJV2003 per mg gold), 0.5 mggold per cartridge;

[0183] Formulation #4: pM2-FL DNA vector, pPJV2006 and pPJV2003 DNAvectors all coprecipitated onto single batch of gold (2 μg pM2-FL DNAper mg gold, and 1 μg each of pPJV2006 and pPJV2003 DNA per mg gold),0.5 mg gold per cartridge;

[0184] Formulation #5: pM2-FL DNA vector and pPJV2002 coprecipitatedonto single batch of gold (2 μg pM2-FL DNA per mg gold, and 1 μg ofpPJV2002 DNA per mg gold), 0.5 mg gold per cartridge; and

[0185] Formulation #6: pM2-FL DNA vector and pPJV2003 coprecipitatedonto single batch of gold (2 μg pM2-FL DNA per mg gold, and 1 μg ofpPJV2003 DNA per mg gold), 0.5 mg gold per cartridge.

[0186] These DNA vaccine formulations were then administered to sixgroups of mice as follows. Each experimental group contained 7 animalsand each animal received two immunizations with the respectiveformulation with a 4 week resting period between immunizations. Eachimmunization consisted of two tandem deliveries to the abdominalepidermis (one cartridge per delivery) using a PowderJect® XR-1 particledelivery device (PowderJect Vaccines Inc., Madison, Wis.) at a heliumpressure of 400 p.s.i.. Serum samples were collected 4 weeks followingthe primary immunization Oust before the boost) and two weeks followingthe second or booster immunization.

[0187] Individual serum samples were assayed for M2-specific antibodyresponses using an ELISA assay in which 96-well plates were pre-coatedwith an M2 synthetic peptide consisting of the following sequence:SLLTEVETPIRNEWECR (SEQ ID NO: 19). ELISA plates were coated with the M2peptide overnight at 4° C. using the peptide in phosphate bufferedsaline (PBS) at a concentration of 1 μg/ml. On the next day, the plateswere blocked with 5% nonfat dry milk in PBS for 1 hour at roomtemperature. Plates were then washed three times with wash buffer (10 mMTris Buffered Saline, 0.1% Brij-35). Diluted serum samples were thenadded to the wells and the plates were incubated for 2 hours at roomtemperature. The plates were washed three times with wash buffer and 100μl of a secondary antibody was added and plates were incubated for 1hour at room temperature. The secondary antibody consisted of a goatanti-mouse IgG (H+L) biotin-labeled antibody (Southern Biotechnology)that was diluted 1:8000 in 1% BSA/PBS/0.1% Tween-20. Plates were thenwashed three times, and a streptavidin-horse radish peroxidase conjugate(Southern Biotechnology) diluted to 1:8000 in PBS/0.1% Tween-20 wasadded and the plate incubated for 1 hour at room temperature. Followingthree additional washes, 100 μl of TMB substrate (Bio Rad, Hercules,Calif.) was added and color development was allowed to proceed for 30minutes at room temperature. Color development was stopped by theaddition of 1N H₂SO₄ and the plates were read of 450 nm. Endpointdilution titers were determined by identifying the highest dilution ofserum that still yielded an absorbance value that was two times thebackground absorbance value obtained using a non immune control sample.

[0188] Endpoint antibody titers for individual animals in each group andgeometric mean titers for each experimental group are shown in Table 1below. TABLE 1 Formulation # Individual Titers Geometric Mean Titer 124,300 99,781 24,300 72,900 218,700 218,700 218,700 218,700 2 72,900186,934 72,900 218,700 218,700 218,700 218,700 656,100 3 24,300 186,93472,900 72,900 218,700 656,100 656,100 656,100 4 24,300 350,211 218,700656,100 656,100 656,100 656,100 656,100 656,100 5 —* 262,645 72,900218,700 218,700 218,700 656,100 656,100 6 24,300 409,722 72,900 218,700218,700 1,968,300 1,968,300 5,904,900

[0189] As can be seen, all experimental groups immunized with aformulation containing one or more of the CT-encoding adjuvant vectors(Formulations #2-6) exhibited an increased geometric mean titerfollowing the booster immunization relative to control animals immunizedwith the M2 vector (Formulation #1) alone.

Example 4 Enhancement of Antigen-Specific Cellular Responses to a DNAVaccine Encoding the Hepatitis B Surface Antigen (HBsAg) Using AdjuvantPlasmid Vectors Encoding the CT-A and CT-B Subunit Peptides

[0190] A Hepatitis B surface antigen (HBsAg) vector plasmid wasconstructed as follows. To generate the HbsAg coding region, the pAM6construct (obtained from the American Type Culture Collection “ATCC”)was cut with NcoI and treated with mung bean nuclease to remove thestart codon of the X-antigen. The resultant DNA was then cut with BamHIand treated with T4 DNA polymerase to blunt-end the DNA and create anHBsAg expression cassette. The HBsAg expression cassette is present inthe 1.2 kB fragment. The plasmid construct pPJV7077 (Schmaljohn et al.(1997) J. Virol. 71:9563-9569) which contains the full-length human CMV(Towne strain) immediate early promoter (with enhancer) was cut withHindIII and BgIII, and then treated with T4 DNA polymerase andcalf-alkaline phosphatase to create blunt-ended DNA, and the HBsAgexpression cassette was ligated into the plasmid to yield the pWRG7128construct.

[0191] The pWRG7128 plasmid was precipitated onto gold particlesfollowing the procedure described in Example 3 above, again using 2 μgDNA per mg gold. Adjuvant plasmid vectors expressing the CTA and CTBsubunit genes (pPJV2002 and pPJV2003, respectively) were mixed togetherand precipitated onto gold particles with each plasmid being present at1 μg DNA per mg gold such that the total amount was also 2 μg DNA per mggold. The gold particles coated with pWRG7128 and those coated with thepPJV2002 and pPJV2003 were mixed 1:1 and then loaded into TEFZEL® tubingas above. For immunization, 0.5 inch lengths of tubing representing 1 μgDNA (0.5 μg pWRG7128 plasmid and 0.25 μg of each of pPJV2002 andpPJV2003 plasmid) were delivered into the epidermis of Balb/c mice usingthe PowderJect® XR-1 particle delivery device using the same deliveryconditions as described above in Example 3. For controls, mice wereimmunized with gold particles coated only with pWRG7128 (1 μg DNA/0.5 mggold per delivery). Mice (4 per experimental group) were immunized andboosted at 4 weeks, then sacrificed at 2 weeks post-boost. Immuneresponses were evaluated for serum antibody levels by ELISA. Inaddition, cellular immune responses were measured using an ELISPOT assayto quantify CD8-specific IFN-γ secretion.

[0192] Serum samples of individual mice were tested for antibodiesspecific for HBsAg using an ELISA assay. For the ELISA, Falcon Pro Bindmicrotiter plates were coated overnight at 4° C. with purified HBsAg(BioDesign) at 0.1 μg per well in PBS (phosphate buffered saline,BioWhittaker). The plates were blocked for 1 hour at room temperature(RT) with 5% dry milk/PBS then washed 3 times with wash buffer (10 mMTris Buffered saline, 0.1% Brij-35), and serum samples diluted indilution buffer (2% dry milk/PBS/0.05% Tween 20) were added to the plateand incubated for 2 hours at RT. The plates were washed 3 times and abiotinylated goat anti-mouse antibody (Southern Biotechnology) diluted1:8000 in dilution buffer was added to the plate and incubated for 1 hrat RT. Following the incubation, plates were washed 3 times, after whicha Streptavidin-Horseradish peroxidase conjugate (Southern Biotechnology)diluted 1:8000 in PBS was added and the plate incubated a further 1 hrat RT. After an additional three washes, Plates were washed 3 times,then a TMB substrate solution (BioRad) was added and the reaction wasstopped with 1N H₂SO₄ after 30 minutes. Optical density was read at 450nm. Endpoint titers were calculated by comparison of the samples with astandard of known titer.

[0193] For the cellular immune assays, single cell suspensions ofsplenocytes from the spleens of the immunized animals were cultured invitro in the presence of a peptide corresponding to a known CD8 epitopein Balb/c mice. The peptide was dissolved in DMSO (10 mg/ml) and dilutedto 10 ug/ml in culture. The sequence of the peptide was IPQSLDSWWTSL(SEQ ID NO: 20).

[0194] For IFN-γ ELISPOT assays, Millipore Multiscreen membranefiltration plates were coated with 50 μl of 15 μg/ml anti-IFN-γantiserum (Pharmingen) in sterile 0.1 M carbonate buffer (pH 9.6)overnight at 4° C. Plates were washed 6 times with sterile PBS and thenblocked with tissue culture medium containing 10% fetal bovine serum(FBS) for 1-2 hr at RT. The medium was removed and spleen cellsdispensed into the wells with a total of 1×10⁶ cells per well. For wellsin which less than 1×10⁶ cells from immunized animals was added, cellsfrom naïve animals were used to bring the total to 1×10⁶. Cells wereincubated overnight in a tissue culture incubator in the presence of thepeptide as described above. The plates were then washed 2 time with PBSand 1 time with distilled water. This was followed by 3 washes with PBS.A biotinylated anti IFN-γ monoclonal antibody (Pharmingen) was added tothe plate (50 μl of a 1 μg/ml solution in PBS) and incubated for 2 hr atRT. The plates were washed 6 times with PBS after which 50 μl of aStreptavidin Alkaline phosphatase conjugate (1:1000 in PBS, Pharmingen)was added and incubated for 2 hr at RT. The plates were washed 6 timeswith PBS and an alkaline phosphatase color substrate (BioRad) was addedand the reaction was allowed to proceed until dark spots appeared. Thereaction was stopped by washing with water 3 times. Plates were airdried and spots counted under a microscope.

[0195] For the IFN-γ ELISA assays, the cells were cultured overnight inround bottom 96 well tissue culture plates in the presence of thepeptide. Samples of the supernatant were taken and used for thedetermination of IFN-γ levels. High binding plates (Costar) were coatedwith 100 μl of 0.5 ug/ml of anti-mouse IFN-γ antibody (Pharmingen) inbicarbonate buffer pH 9.6. Plates were blocked for 1 hr at RT withtissue culture medium containing 10% FBS then washed 3 times with a TBSwash buffer. Supernatant samples obtained from cultured cells werediluted in tissue culture medium and loaded onto the plate and incubatedfor 2 hr at RT. Plates were washed 3 times with wash buffer and asecondary antibody (0.5 μg/ml of biotinylated rat anti-mouse INF-γ inPBS, Pharmingen) was added to the plates and incubated for 1 hr at RT.Plates were washed 3 times, and a Streptavidin-horseradish peroxidaseconjugate (1:2000 in PBS, Southern Biotechnology) was added for 1 hr atRT. Plates were washed 3 times, then a TMB substrate solution was added(BioRad) and the reaction was stopped with 1N H₂SO₄. Optical density wasread at 450 nm.

[0196] The results of the ELISA are reported below in Table 2. TABLE 2Formulation Individual Titers Average Titers pWRG7128 40,000 55,00084,000 41,000 54,000 pWRG7128, 28,000 23,000 pPJV2002 and 14,000pPJV2003 27,000 23,000

[0197] As can be seen, antibody levels in control mice immunized withpWRG7128 alone were found to be higher than in those immunized withpWRG7128 in combination with the CT adjuvant vectors (pPJV2002 andpPJV2003). The average endpoint titer for the control animals was55,000, while the average titer in the animals immunized with theadjuvanted formulation was 23,000. However, the group receiving theadjuvanted formulation actually received ½ the amount of pWRG7128 as thecontrols, which may account for the reduction in antibody titers.

[0198] The cellular immune responses measured in the two groups of miceindicate a significant enhancement of cellular responses by the CTadjuvant vectors. More particularly, the results of the CD8-specificIFN-γ ELISA assay are depicted below in Table 3. TABLE 3 IFN-γ ELISANumber of Cells/Well Formulation 1.0 × 10⁶ 0.5 × 10⁶ 0.1 × 10⁶ pWRG71280.77 0.213 0.001 0.828 0.121 0.027 1.35 0.312 0.006 1.25 0.323 0.007(Average) 1.05 0.242 0.01 pWRG7128, 1.96 1.19 0.079 pPJV2002 and 2.301.70 0.263 pPJV2003 2.20 1.83 0.377 2.44 2.33 0.898 (Average) 2.23 1.760.404

[0199] In this IFN-γ ELISA assay, the number of cells per wellrepresents the number of cells recovered from immunized animals platedper well. Total number of cells per well is constant (e.g., 1×10⁶) andis supplemented with cells from naïve animals. Values are the ODreadings measured at 450 nm. The higher OD values in the ELISA found forthe cells from mice treated with the CT adjuvanted formulation isindicative of a greater amount of IFN-γ secreted by these cells inresponse to antigen. Cells from naive mice did not yield a measurable ODvalue in the IFN-γ ELISA.

[0200] The results of the CD8-specific IFN-γ ELISPOT assay are depictedbelow in Table 4. TABLE 4 IFN-γ ELISPOTs Formulation Number of PositiveCells/1 × 10⁶ Cells pWRG7128 510 410 530 590 (Average) 510 pWRG7128,1,480 pPJV2002 and 2,300 pPJV2003 2,500 3,500 (Average) 2,445

[0201] In this IFN-γ ELISPOT assay, the numbers are the average ofduplicate wells and correspond to number of positive cells per 1×10⁶cells. Cells from naïve mice did not yield any spots in this ELISPOTassay. In addition, the greater number of ELISPOTs found in the animalstreated with the CT adjuvanted formulation indicates a superior responseas compared with the animals receiving the antigen (pWRG7128) alone.

[0202] The above data demonstrate that the novel adjuvant compositionsof the present invention have a potent ability to enhance the cellularimmune responses to a coadministered antigen, in this case a HBsAgexpressed from a DNA vaccine.

Example 5 Enhancement of Humoral and Cellular Immune Responses to HIV-1gp120 Antigen Using Simultaneous Delivery of a gp120 Antigen Vector andCTA/CTB Adjuvant Vectors

[0203] A plasmid vector endoding HIV-1 gp120 was constructed as follows.The vector was constructed starting with a Bluescript (Stratagene, LaJolla, Calif.) plasmid backbone, the human cytomegalovirus (hCMV)immediate early promoter (Fuller et al. (1994) Aids Res. HumRetroviruses 10:1433) and the SV40 virus late polyadenylation site. ThehCMV promoter is contained within a 619 base pair (bp) AccII fragmentextending 522 bp upstream and 96 bp downstream from the immediate earlytranscription initiation site. The SV40 virus late polyadenylationsequence is contained within an approximately 800 bp BamHI-BglIIfragment derived from pSV2dhfr (formerly available from BethesdaResearch Laboratories, catalogue #5369 SS). Initially, a plasmidencoding HIV-1 gp160, termed “pC-Env” was constructed. This plasmidcontains a 2565 bp KpnI-XhoI fragment from LAV-1_(BRU) (ATCC AccessionNo. 53069, GenBank Accession No. K02013), which begins at the sequenceencoding amino acid position #4 of the mature gp160 amino terminus. Theenv coding sequence fragment was placed immediately downstream of, andfused in frame with a 160 bp synthetic fragment encoding the herpessimplex virus glycoprotein D (gD) signal peptide and none amino acids ofthe mature gD amino terminus as previously described (Fuller et al.(1994) Aids Res. Hum Retroviruses 10:1433).

[0204] The plasmid encoding HIV-1 gp120, termed “pCIA-Env/T” herein, wasthen constructed as follows. The pCIA-Env/T plasmid encodes a truncatedform of HIV-1 gp160, and is identical to the pC-Env construct exceptthat the env coding sequences are truncated at the HindIII site atnucleotide position 8188. This results in a truncated gp160 translationproduct with the truncation point lying 128 amino acid residuesdownstream of the gp120/gp41 processing site.

[0205] A second plasmid vector encoding HIV-1 rev, termed “pC-rev”herein, was constructed as follows. This vector contains threediscontinuous regions of the LAV-1_(BRU) provirus (nucleotide positions678-1085, 5821-6379, and 8188-8944) placed directly between the hCMVpromoter and the SV40 virus late polyadenylation sequence as describedabove. The three discontinuous regions contain the major 5′ splicingdonor, the first exon of the rev gene, and the second exon of the revgene, respectively.

[0206] A third plasmid vector construct termed “pWRG7054” was used as anempty vector control in the study. The pWRG7054 construct contains anSIV nef coding region, the hCMV promoter immediate early with the IntronA region, a TPA leader sequence and the bovine growth hormonepolyadenylation sequence. Construction of the pWRG7054 plasmid isdescribed herein below in Example 6.

[0207] The following DNA-gold formulations were generated for a mouseDNA vaccine adjuvant trial.

[0208] Formulation #1: Empty vector control (pWRG7054 without the gp120insert), 2.5 μg DNA per mg gold, 0.5 mg gold per cartridge;

[0209] Formulation #2: Empty vector control (pWRG7054), the gp120(pCIA-EnvT) DNA vector, and the rev (pC-rev) DNA vector (to allow forexpression of the HIV-1 gp120 molecule), all coprecipitated onto asingle batch of gold (1.25 μg of each of pWRG7054 DNA and pCIA-EnvT DNAper mg gold, and 0.125 μg of pC-rev per mg gold), 0.5 mg gold percartridge;

[0210] Formulation #3: Empty vector control (pWRG7054), CTA (pPJV2002)and CTB (pPJV2003) DNA vectors all coprecipitated onto a single batch ofgold (1.25 μg pWRG7054 DNA per mg gold, and 1 μg each of pPJV2002 andpPJV2003 DNA per mg gold), 0.5 mg gold per cartridge;

[0211] Formulation #4: gp120 (pCIA-EnvT) DNA vector, HIV-1 rev (pC-rev)DNA vector, CTA (pPJV2002) and CTB (pPJV2003) DNA vectors allcoprecipitated onto a single batch of gold (1.25 μg pCIA-EnvT DNA per mggold, 0.125 μg pC-rev DNA per mg gold, and 1 μg each of pPJV2002 andpPJV2003 DNA per mg gold), 0.5 mg gold per cartridge; and

[0212] Formulation #5: gp120 (pCIA-EnvT) DNA vector, HIV-1 rev (pC-rev)DNA vector, CTA-KDEL (pPJV2006) and CTB (pPJV2003) DNA vectors allcoprecipitated onto a single batch of gold (1.25 μg pCIA-EnvT DNA per mggold, 0.125 μg pC-rev DNA per mg gold, and 1 μg each of pPJV2006 andpPJV2003 DNA per mg gold), 0.5 mg gold per cartridge.

[0213] The pWRG7054, pCIA-EnvT, pC-rev, pPJV2002, pPJV2003 and pPJV2006plasmid vectors were precipitated onto gold particles following theprocedures described in Example 3 above, again using 2 μg DNA per mggold. The coated gold particles were loaded into TEFZEL® tubing, againusing the procedures described in Example 3 above. For immunization, two0.5 inch lengths of tubing were used to deliver a total payload of 1.0mg gold into the epidermis of 5-6 week old female Balb/c mice using aparticle delivery device operated under the same delivery conditions asdescribed above in Example 3 (400 p.s.i. helium).

[0214] Each of the five experimental groups (one per each DNA vaccineformulation) consisted of 4 animals, and each animal received primaryand booster immunizations with their respective formulations, timed at 0and 5 weeks. Each immunization consisted of two tandem deliveries to theabdominal epidermis (one cartridge per delivery) using a PowderJect®XR-1 particle delivery device (PowderJect Vaccines Inc., Madison, Wis.).

[0215] Serum antibody responses to the HIV gp120 antigen were testedusing an ELISA assay on specimens collected at week 5 and week 6.5(post-prime and post-boost, respectively). For the ELISA, Costar highbinding EIA plates were coated with 0.3 μg/well of recombinant HIV gp120(Intracel) in 50 μl PBS by incubation overnight at 4° C. Plates werewashed three times and blocked with 2% BSA in PBS for 2 hours at roomtemperature. Serial dilutions of serum were added to the coated plates,and incubated at 37° C. for one hour. After washing, the plates wereincubated with a 1:1500 dilution of alkaline phosphatase conjugated goatanti-mouse IgG (H+L) (BioRad), followed by color development withp-nitrophenylphosphate (PNPP) (BioRad) and OD reading @ 405 nm.

[0216] The results of the ELISA are depicted in FIG. 7. As can be seen,there was an approximate 20-fold augmentation of gp120-specific immuneresponses in the groups that received adjuvant vectors (Formulation #4containing the pPJV2002 and pPJV2003 vectors, or Formulation #5containing the pPJV2006 and pPJV2003 vectors) in combination with thegp120 vector as compared with the group that received the gp120 vectorwithout adjuvant (Formulation #2, pCIA-EnvT).

[0217] Following collection of the post-boost serum samples, animalswere sacrificed and spleens were collected from each mouse. Splenocyteswere isolated by crushing the spleens, passing the cells through a 70 μmcell strainer, and lysing the red blood cells with ACK lysis buffer(BioWhittaker). Splenocytes were washed 3 times with RPMI-5% FCS andresuspended to a concentration of 1×10⁷ cells/ml in RPMI-10% FCSsupplemented with antibiotics, sodium pyruvate, and non-essential aminoacids.

[0218] The amount of antigen-specific IFN-γ secreted by the splenocyteswas determined using an in situ ELISA. Costar high binding plates werecoated with 10 μg/ml of anti-mouse IFN-gamma capture monoclonal antibody(mAb) (Pharmingen, San Diego, Calif.) in 50 μl 0.1M bicarbonate buffer(pH 9.6). After overnight incubation at 4° C., the wells were washed 5times with PBS-0.05% Tween-20 and blocked with 200 μl of complete RIOmedium at room temperature for 2 hours. 1×10⁶ splenocytes were added toeach well and were stimulated in medium alone (negative control), or inmedium with 1 μg/ml of a HIV gp120 peptide having the followingsequence: RIQRGPGRAFVITGK (SEQ ID NO: 21). Following a 24 hourincubation at 37° C. in 5% CO₂, the plates were washed 2 times withdeionized (DI) water to lyse the cells, then washed 3 times withPBS-0.05% Tween 20, and incubated for 1 hour at room temperature with 50μl per well of 1 μg/ml biotinylated anti-mouse IFN-γ detecting mAb(Pharmingen). The plates were then washed 5 times and incubated for 1hour with 50 μl per well of a 1:8000 dilution of strepavidin-horseradishperoxidase (HRP) solution (Southern Biotechnology). The plates wereagain washed 5 times and colorimetric development was accomplished bythe addition of TMB substrate (BioRad, Hercules, Calif.) for 30 minutesat room temperature. The reaction was stopped by the addition of INsulfuric acid. Absorbance at 450 nm was read with an optical platereader.

[0219] The results of this study are depicted in FIG. 8. This figureshows the relative level of antigen-specific IFN-γ production in the 5different vaccine test groups receiving the above-described formulations(Formulations #1-5). Importantly, the two immunization groups thatreceived the gp120 vector (pCIA-EnvT) in combination with the CTadjuvant vectors (i.e., Formulation #4 containing the pPJV2002 andpPJV2003 vectors, and Formulation #5 containing the pPJV2006 andpPJV2003 vectors) displayed significantly higher IFN-γ production levelsthan did the gp120 without adjuvant group (Formulation #2 containing theempty vector control and pCIA-EnvT), (P<0.000001 and P=0.0068,respectively). These data demonstrate the ability of the present CTvector adjuvant combinations to markedly augment antigen-specificcellular immunity to an HIV antigen in an animal model.

[0220] In addition to an increase in the level of IFN-γ production, thenumber of IFN-γ secreting HIV-gp120 peptide-specific splenocytes wasalso increased markedly, as determined by an ELISPOT method. In theELISPOT assay, nitrocellulose plates (Millipore) were coated with anIFN-γ capture antibody, washed, and blocked as described above for the Thelper cell IFN-γ in situ ELISA. Splenocytes were added to pre-coatedwells at an input cell number of 1×10⁶ cells/well, and stimulated inmedium alone (negative control) or in medium containing 1 μg/ml of apeptide containing the immunodominant HIV-gp120 CTL epitope and havingthe following sequence: RGPGRAFVTI (SEQ ID NO: 22). The plates wereincubated for 24 hours at 37° C. in 5% CO₂, washed 2 times with DIwater, 3 times with PBS, and incubated for 1 hour at room temperaturewith 50 μl per well of 1 μg/ml biotinylated anti-mouse IFN-γ detectingmAb (Pharmingen). The plates were then washed 5 times and incubated for1 hour with 50 μl per well of a 1:1000 dilution of strepavidin-alkalinephosphatase (ALP) solution (Mabtech). The plates were again washed 5times and colorimetric development was accomplished by the addition ofALP membrane substrate (BioRad, Hercules, Calif.) until spots formed(2-30 minutes, room temperature). The reaction was stopped by washingwith DI water, and the plates were air-dried overnight. Spots wereenumerated under a 40× microscope. Only large spots with fuzzy borderswere scored as spot forming cells (SFC).

[0221] The results from this ELISPOT assay are depicted in FIG. 9 whichshows the relative levels of IFN-γ-producing splenocytes in the 5different vaccine test groups (receiving Formulations #1-5,respectively). Importantly, the two immunization groups that receivedthe gp120 vector in combination with the CT adjuvant vectors (i.e.,Formulation #4 containing the pPJV2002 and pPJV2003 vectors, andFormulation #5 containing the pPJV2006 and pPJV2003 vectors) displayedsignificantly higher numbers of IFN-γ-producing cells than did the gp120without adjuvant group (Formulation #2 containing the empty vectorcontrol and pCIA-EnvT), (P<0.000001 and P=0.0032, respectively). Thesedata demonstrate the ability of the present CT adjuvant combinations tomarkedly augment antigen-specific cellular immunity against acoadministered HIV gp120 antigen in an animal model. In addition, theenhanced IFN-γ production seen in these studies indicates that use ofthe CT adjuvant vector combinations of the present invention provides arobust Th1-like immune response in the immunized animals.

Example 6 Enhancement of Antibody Responses to Hepatitis B Core andSurface Antigens Using Simultaneous Delivery of a Vector Encoding HBcAgand HBsAg with CTA/CTB Adjuvant Vectors

[0222] A vector plasmid containing coding sequences for both theHepatitis B core antigen (HBcAg) and Hepatitis B surface antigen (HBsAg)was constructed as follows. The HBcAg and HBsAg coding sequences wereboth obtained from the HBV clone pAM6 (ATCC Accession No. 45020). Togenerate the HBsAg coding region, the pAM6 construct was cut with NcoIand treated with mung bean nuclease to remove the start codon of theX-antigen. The resultant DNA was then cut with BamHI and treated with T4DNA polymerase to blunt-end the DNA and create an HBsAg expressioncassette. The HBsAg expression cassette is present in the 1.2 kBfragment. The plasmid construct pPJV7077 (Schmaljohn et al. (1997) J.Virol. 71:9563-9569) which contains the full-length human CMV (Townestrain) immediate early promoter (with enhancer) was cut with HindIIIand BglII, and then treated with T4 DNA polymerase and calf-alkalinephosphatase to create blunt-ended DNA, and the HBsAg expression cassettewas ligated into the plasmid to yield the pWRG7128 construct.

[0223] To generate the HBcAg coding region, the pAM6 construct was cutto create an HBcAg expression cassette, after which the HBcAg sequencewas truncated by site directed mutagenesis to remove the C-terminalarginine-rich region from the core antigen particle (which deletion doesnot interfere with particle formation). The truncated HBcAg sequence wasthen cloned into a plasmid construct containing the human elongationfactor promoter (“hELF”, Mizushima et al. (1990) Nucl. Acids Res.18:5322) to provide a HBcAg vector construct.

[0224] Expression cassettes containing: (a) the CMV promoter/enhancer,the Intron A-5′ untranslated region, and the human tissue plasminogenactivator (hTPA) signal peptide (“CMV-IA-TPA”); or (b) the bovine growthhormone polyA sequence (bGHpA) were each obtained from the JW4303 vectorconstruct (gift of Dr. Harriet Robinson, University of Massachusetts)and inserted into a plasmid backbone. The resultant construct was cutwith NheI, filled with polymerase and then cut with BamHI to generate avector fragment containing the pUC19 origin of replication, theampicillin resistance gene and the bGHpA sequence. The plasmid backbonewas cut a second time with SalI, filled with polymerase, and cut withBamHI to liberate a vector fragment containing the CMV-IA-TPA vectorfragment. The two vector fragments were ligated together to yield aconstruct termed pWRG7054.

[0225] The pWRG7054 construct was cut with NheI, filled with polymerase,and cut with BamHI to produce a vector fragment. The HBcAg vectorconstruct was cut with NcoI, filled with polymerase, and cut with BamHIto produce an insert fragment. The two fragments were then ligatedtogether to yield a construct termed pWRG7063.

[0226] PEL-Bos was cut with EcoRI and dephosphorylated with calfintestinal phosphatase to produce a vector fragment. The pWRG7063plasmid was cut with HindIII, filled with polymerase, and cut with EcoRIto produce an insert fragment containing the hTPA signal peptide, theHBcAg antigen sequence and the bGHpA region. These two fragments wereligated together to provide a construct termed pWRG7145.

[0227] The pWRG7128 construct was cut with EcoRI and dephosphorylatedwith calf intestinal phosphatase to produce a vector fragment containingthe HBsAg coding region under transcriptional control of the hCMVpromoter. The pWRG7145 construct was cut with MfeI and EcoRI to producean insert fragment comprised of the hELF promoter/intron, the hTPAsignal peptide sequence, the HBcAG antigen sequence and the bGHpAregion. These fragments were then ligated together to provide thepPJV7193 plasmid construct containing the HBcAg and HBsAg codingsequences.

[0228] The following DNA-gold formulations were then generated for a pigDNA vaccine adjuvant trial.

[0229] Formulation #1: Control, the HBcAg/HBsAg vector (pWRG7193) alone,2 μg DNA per mg gold, 0.5 mg gold per cartridge; and

[0230] Formulation #2: The HBcAg/HBsAg vector (pWRG7193), CTA-KDEL(pPJV2006) and CTB (pPJV2003) DNA vectors all coprecipitated onto singlebatch of gold (1.0 μg pWRG7193 DNA per mg gold, and 0.5 μg each ofpPJV2006 and pPJV2003 DNA per mg gold), 0.5 mg gold per cartridge.

[0231] The pWRG7193 plasmid either alone or with the pPJV2006 andpPJV2003 adjuvant vectors was precipitated onto gold particles followingthe procedures described in Example 3 above, again at a finalconcentration of 2 μg DNA per mg gold. The coated gold particles wereloaded into TEFZEL® tubing using the procedures described in Example 3above. Two experimental groups (of 5 domestic pigs each) received twoimmunizations with Formulation #1 and Formulation #2, respectively,wherein the two immunizations were spaced 6 weeks apart. Eachimmunization consisted of two tandem 500 p.s.i. shots to the groin areausing a PowderJect® XR-1 particle delivery device in which each shotutilized a single cartridge. Thus, in the control group (Formulation#1), each immunization consisted of the delivery of a total of 1 mg goldand 2 μg of the DNA vaccine vector pWRG7193. Similarly, in the adjuvanttest group (Formulation #2), each immunization consisted of a totaldelivery of 1 mg of gold, 2 μg of the DNA vaccine vector pWRG7193, and 1μg each of the adjuvant vectors pPJV2006 and pPJV2003. Blood sampleswere collected from each animal at the time of the booster immunization(week 6) and 2 weeks following the booster immunization (week 8).

[0232] Detection of antibody responses specific for the core antigen wasperformed as follows: ELISA plates were coated with the hepatitis B coreantigen (Biodesign) at 100 ng/ml in PBS. After coating overnight at 4°C., plates were blocked with 5% nonfat dry milk in PBS for 1 hour atroom temperature. Plates were then washed 3 times with PBS containing0.05% Tween-20. Swine serum samples were serially diluted in 2% nonfatdry milk/PBS/0.01% Tween-20 and added to the ELISA plates. Afterincubation at room temperature for 2 hours, plates were washed 3 timeswith PBS/0.05% Tween-20. The secondary antibody consisted of a goatanti-swine IgG conjugated to horse radish peroxidase (Kirkegaard andPerry) that was diluted 1:2000 in 2% nonfat dry milk/PBS/0.01% Tween-20,and added to the plates for a 1 hour incubation at room temperature.Plates were then washed 5 times with PBS/0.05% Tween-20 and 100 μl ofTMB substrate as added. Color development proceeded for 15 minutes andwas stopped by the addition of 100 μl of 1N H₂SO₄. Plates were read at450 nm. FIGS. 10 and 11 show the geometric mean absorbance values at 6and 8 weeks, respectively, for each of the two groups of pigs at 4different serum dilutions. These data demonstrate marked enhancement ofantibody titers to the hepatitis B core antigen following use of CTadjuvant vectors pPJV2006 and pPJV2003.

[0233] In addition to elevated humoral responses to the hepatitis B coreantigen, a measurable elevation of antibody responses specific for thehepatitis B surface antigen was observed in the adjuvant test group aswell. Surface antigen-specific antibodies were quantified in serumsamples from individual animals using a commercial assay kit (AUSAB,Abbott Laboratories). This kit allows for the quantification of antibodyresponses in terms of milli-international unit (mIU/ml) using a standardpanel. The geometric mean surface antigen antibody titers in the controlgroup (Formulation #1) and adjuvant test group (Formulation #2) were 285and 662 mIU/ml, respectively, demonstrating the ability of the presentadjuvant plasmids (pPJV2006 and pPJV2003) to augment immune responses toan antigen encoded by a separate vector.

Example 7 Enhancement of Cellular Th1-Like Immune Response to a DNAVaccine Using Plasmid Vectors Encoding CT or LT Subunits

[0234] The pM2-FL DNA vaccine vector encoding the M2 protein ofinfluenza A virus was employed to test the adjuvant effects of thepPJV2002, pPJV2003, pPJV2004, pPJV2005, pPJV2006 and pPJV2007 adjuvantvectors in the context of particle-mediated DNA vaccination.Particle-mediated DNA vaccination was performed by precipitating the M2DNA vaccine vector, either with our without various combinations of theadjuvant vectors, onto microscopic gold particles and accelerating thecoated gold particles into the epidermis of mice using a PowderJect®XR-1 particle delivery device (PowderJect Vaccines, Inc. Madison, Wis.).Construction of the pM2-FL DNA plasmid vector is described herein abovein Example 3.

[0235] As above, the pM2-FL plasmid was precipitated onto 2 micron goldparticles as single vector, or mixed vector plus adjuvant vectorsamples. Specifically, plasmid DNA (single pM2-FL vector or pM2-FLvector plus one or more adjuvant vectors) was mixed with 2 micron goldparticles (Degussa) in a small centrifuge tube containing spermidine.Precipitation was carried out following the methodologies of Example 3,and the coated gold particles were then coated onto the inside surfaceof a TEFZEL® tube as also described in Example 3. The tubing was thencut into 0.5 inch cartridges suitable for loading into the particledelivery device.

[0236] The following DNA-gold formulations were generated for a mouseDNA vaccine adjuvant trial.

[0237] Formulation #1: pM2-FL DNA vector combined with the pWRG7054empty plasmid vector prior to precipitation onto the same batch of gold,2.1 μg total DNA per mg gold (0.1 μg pM2-FL and 2.0 μg pWRG7054), 0.5 mggold per cartridge;

[0238] Formulation #2: pM2-FL DNA vector combined with the CTA and CTB(pPJV2002 and pPJV2003) DNA vectors prior to precipitation onto a singlebatch of gold, 2.1 μg total DNA per mg gold (1.0 μg of each DNA adjuvantvector per mg gold, 0.1 μg pM2-FL), 0.5 mg gold per cartridge;

[0239] Formulation #3: pM2-FL DNA vector combined with the CTA-KDEL andCTB (pPJV2006 and pPJV2003) DNA vectors all coprecipitated onto singlebatch of gold, 2.1 μg total DNA per mg gold (1.0 μg of each DNA adjuvantvector per mg gold, 0.1 μg pM2-FL), 0.5 mg gold per cartridge;

[0240] Formulation #4: pM2-FL DNA vector combined with the CTA-KDEL(pPJV2006) DNA vector and supplemented with the pWRG7054 empty plasmidvector all combined and coprecipitated onto single batch of gold, 2.1 μgtotal DNA per mg gold (1.0 μg pPJV2006, 1.0 μg pWRG7054, 0.1 μg pM2-FL),0.5 mg gold per cartridge;

[0241] Formulation #5: pM2-FL DNA vector combined with the CTA(pPJV2002) DNA vector and supplemented with the pWRG7054 empty plasmidvector, all DNAs combined and coprecipitated onto single batch of gold,2.1 μg total DNA per mg gold (1.0 μg pPJV2002, 1.0 μg pWRG7054, 0.1 μgpM2-FL), 0.5 mg gold per cartridge;

[0242] Formulation #6: pM2-FL DNA vector combined with the CTB(pPJV2003) DNA vector and supplemented with the pWRG7054 empty plasmidvector, all DNAs combined and coprecipitated onto single batch of gold,2.1 μg total DNA per mg gold (1.0 μg pPJV2003, 1.0 μg pWRG7054, 0.1 μgpM2-FL), 0.5 mg gold per cartridge;

[0243] Formulation #7: pM2-FL DNA vector combined with the LTA and LTB(pPJV2004 and pPJV2005) DNA vectors prior to precipitation onto a singlebatch of gold, 2.1 μg total DNA per mg gold (1.0 μg of each DNA adjuvantvector per mg gold, 0.1 μg pM2-FL), 0.5 mg gold per cartridge;

[0244] Formulation #8: pM2-FL DNA vector combined with the LTA-RDEL andLTB (pPJV2007 and pPJV2005) DNA vectors all coprecipitated onto singlebatch of gold, 2.1 μg total DNA per mg gold (1.0 μg of each DNA adjuvantvector per mg gold, 0.1 μg pM2-FL), 0.5 mg gold per cartridge;

[0245] Formulation #9: pM2-FL DNA vector combined with the LTA-RDEL(pPJV2007) DNA vector and supplemented with the pWRG7054 empty plasmidvector all combined and coprecipitated onto single batch of gold, 2.1 μgtotal DNA per mg gold (1.0 μg pPJV2007, 1.0 μg pWRG7054, 0.1 μg pM2-FL),0.5 mg gold per cartridge;

[0246] Formulation #10: pM2-FL DNA vector combined with the LTA(pPJV2004) DNA vector and supplemented with the pWRG7054 empty plasmidvector, all DNAs combined and coprecipitated onto single batch of gold,2.1 μg total DNA per mg gold (1.0 μg pPJV2004, 1.0 μg pWRG7054, 0.1 μgpM2-FL), 0.5 mg gold per cartridge; and

[0247] Formulation #11: pM2-FL DNA vector combined with the LTB(pPJV2005) DNA vector and supplemented with the pWRG7054 empty plasmidvector, all DNAs combined and coprecipitated onto single batch of gold,2.1 μg total DNA per mg gold (1.0 μg pPJV2005, 1.0 μg pWRG7054, 0.1 μgpM2-FL), 0.5 mg gold per cartridge.

[0248] These DNA vaccine formulations were then administered to elevengroups of mice as follows. Each experimental group contained 8 animalsand each animal received two immunizations with their respectiveformulation with a 4 week resting period between immunizations. Eachimmunization consisted of two tandem deliveries to the abdominalepidermis (one cartridge per delivery) using a PowderJect® XR-1 particledelivery device (PowderJect Vaccines Inc., Madison, Wis.) at a heliumpressure of 400 p.s.i.. Serum samples were collected two weeks followingthe second or booster immunization.

[0249] Individual serum samples were assayed for M2-specific antibodyresponses for both IgG1 and IgG2a subclasses using the ELISA assay ofExample 3 above for the determination of total IgG titer, except that asecondary antibody conjugate specific for either IgG1 or IgG2a wasemployed. The goat anti-mouse IgG1-biotin conjugated antibody wasobtained from Southern Biotechnology Associates, Inc. (catalogue#1070-08, 0.5 mg/ml concentration) and used at a 1/8000 dilution. Thegoat anti-mouse IgG2a-biotin conjugated antibody was obtained from thesame source (catalogue #1080-08, 0.5 mg/ml concentration) and was alsoused at a 1/8000 dilution.

[0250] Geometric mean antibody titers for M2 antigen-specific IgG1 andIgG2a were determined for each experimental group, and the IgG1-to-IgG2aratios were calculated. These data are reported below in Table 5. TABLE5 Formulation # IgG1-to-IgG2a Ratio 1 160.99 2 3.44 3 13.59 4 63.20 52.63 6 30.97 7 0.02 8 0.50 9 9.00 10 4.53 11 27.00

[0251] As can be seen with reference to Table 5, addition of either ofthe A or B subunits, or the various combinations of A and B subunits tothe M2 DNA vaccine formulation resulted in significant reductions in theIgG1-to-IgG2a ratio otherwise elicited with the M2 DNA vaccine (pM2-FL)in the absence of adjuvant (Formulation #1). The greatest ratioreduction resulted from use of the LTA plus LTB, and LTA-RDEL plus LTBvector combinations (Formulations #7 and #8, respectively). In both ofthese formulations, use of the polynucleotide adjuvants of the presentinvention resulted in an abundance of M2 antigen-specific IgG2a titersover IgG1, which characteristic is a hallmark of a Th1-like immuneresponse in mice.

[0252] The results from experimental groups receiving Formulations #1,#2 and #7 were plotted as the log IgG1-to-IgG2a ratio in FIG. 12. As canbe seen in that figure, adjuvanting the pM2-FL DNA vaccine compositionwith the CTA plus CTB adjuvant vectors (Formulation #2) caused a 2 orderof magnitude drop in the IgG1-to-IgG2a ratio relative to the pM2-FLvaccine composition without adjuvant (Formulation #1). In addition, afurther 2 order of magnitude drop in this ratio was observed when thepM2-FL DNA vaccine was adjuvanted with the LTA plus LTB adjuvant vectors(Formulation #7).

Example 8 Addition of Signal Peptide Coding Sequences to AdjuvantVectors Encoding CT or LT

[0253] The vector constructs containing the CTA, CTB, LTA and LTB toxinsubunits (pPJV2002, pPJV2003, pPJV2004, and pPJV2005, respectively) weremodified to remove the tpa signal peptide coding sequences, and a dualDNA vaccine vector encoding both the Hepatitis B surface and coreantigens was constructed for use in the following experiments.

[0254] The standard PCR conditions that were used for theconstruction/modification of the vectors were as follows: 1× PCR corebuffer with 1.5 mM MgCl₂ (Promega Corp., Madison, Wis.); 0.400 μM ofeach primer; 200 μM of each dNTP (USB Inc., Cleveland, Ohio); 2.5 μg Taqpolymerase (Promega Corp., Madison, Wis.); 1.0 ng template DNA; water to100 μl; and a mineral oil (Aldrich Chemical Inc., Milwaukee Wis.)overlay. A PTC-200 thermocycler (MJ Research Inc., Waltham, Mass.) wasprogrammed to run the following routine: 4 minutes @ 95° C.; 30 cyclesof (1 minute @ 95° C./1 minute 15 seconds @ 55° C./1 minute @ 72° C.);10 minutes @ 72° C.; 4° C. hold. The amplification products were removedfrom the PCR reaction using the QIAquick® PCR Purification Kit (QiagenInc., Valencia Calif.) prior to cutting with restriction enzymes (NewEngland Biolabs, Beverly, Mass.). All PCR products were sequenced aftercloning to ensure fidelity of the amplification.

[0255] More specifically, a dual DNA vaccine vector encoding both theHepatitis B surface and core antigens was constructed as follows. ThepWRG7128 construct (Example 4) which contains the surface antigen codingsequence was modified using a series of standard molecular biologytechniques to provide the dual (surface/core antigen) construct.Initially, the pWRG7128 construct was modified to remove the bovinegrowth hormone polyadenylation region and replace the same with therabbit beta-globin polyadenylation region. A first insertion fragmentcontaining the CMV promoter with exon 1 and exon 2 sequences, and asecond insertion fragment containing a second rabbit beta-globinpolyadenlyation region were ligated into the modified pWRG7128construct, and an adaptor constructed by annealing systenticoligonucleotides was inserted between Sph1and Pst1 sites locatedimmediately upstream of the inserted CMV promoter.

[0256] Plasmid mpSmpCC (GlaxoSmithKline, UK) was PCRed with thefollowing primers: 5′-GCC GCT AGC ATG GAC ATT GAC CCT TAT AAA GA-3′ (SEQID NO:23) and 5′-CCA GGA TCC TTA ACA TTG AGA TTC C-3′ (SEQ ID NO:24) togenerate a Hepatitis B-adw2 core antigen coding sequence. This PCRproduct was cut with Nhe1 and BamH1 to generate an insertion fragment,which fragment was then modified to include a downstream Bgl2 site andinserted into a cloning vector. The cloning vector was cut with Pst1 andEcoR1 to generate a core antigen insertion fragment; the modifiedpWRG7128 plasmid was cut with Pst1 and Mfe1 to generate a vectorfragment, and the core antigen insertion fragment was ligated into thevector fragment, resulting in the dual surface/core antigen plasmidconstruct.

[0257] The vector constructs containing the CTA, CTB, LTA and LTB toxinsubunits (pPJV2002, pPJV2003, pPJV2004, and pPJV2005, respectively) weremodified to remove the tpa signal peptide coding sequences by merelyexcising the tpa coding sequences using restriction enzymes to producethe following constructs: CTA w/o TPA, CTB w/o TPA, LTA w/o TPA, and LTBw/o TPA, respectively.

[0258] The dual surface/core antigen plasmid was combined withirrelevant plasmid DNA (for non-adjuvanted control) or with the toxinsubunit vector constructs and precipitated onto 2 micron gold particles.Specifically, plasmid DNA (dual surface/core antigen plasmid vector)plus two adjuvant vectors (to provide a CTA/CTB or LTA/LTB combination)was mixed with 2 micron gold particles (Degussa) in a small centrifugetube containing spermidine. Precipitation was carried out following themethodologies of Example 3, and the coated gold particles were thencoated onto the inside surface of a TEFZEL® tube as also described inExample 3. The tubing was then cut into 0.5 inch cartridges suitable forloading into the particle delivery device.

[0259] The following DNA-gold formulations were thus generated for amouse DNA vaccine adjuvant trial:

[0260] Formulation #1: (“no adjuvant”) 1 μg dual surface/core antigenDNA plasmid vector, 1 μg irrelevant DNA plasmid vector;

[0261] Formulation #2: (“CT”) 1 μg dual surface/core antigen DNA plasmidvector, 0.5 μg pPJV2002 (CTA) DNA plasmid vector, 0.5 μg pPJV2003 (CTB)DNA plasmid vector;

[0262] Formulation #3: (“CT w/o TPA”) 1 μg dual surface/core antigen DNAplasmid vector, 0.5 μg CTA w/o TPA DNA plasmid vector, 0.5 μg CTB w/oTPA DNA plasmid vector;

[0263] Formulation #4: (“CTA”) 1 μg dual surface/core antigen DNAplasmid vector, 1 μg pPJV2002 (CTA) DNA plasmid vector;

[0264] Formulation #5: (“CTA w/o TPA”) 1 μg dual surface/core antigenDNA plasmid vector, 1 μg CTA w/o TPA DNA plasmid vector;

[0265] Formulation #6: (“CTB”) 1 μg dual surface/core antigen DNAplasmid vector, 1 μg pPJV2003 (CTB) DNA plasmid vector;

[0266] Formulation #7: (“CTB w/o TPA”) 1 μg dual surface/core antigenDNA plasmid vector, 1 μg CTB w/o TPA DNA plasmid vector;

[0267] Formulation #8: (“LT”) 1 μg dual surface/core antigen DNA plasmidvector, 0.5 μg pPJV2004 (LTA) DNA plasmid vector, 0.5 μg pPJV2005 (LTB)DNA plasmid vector;

[0268] Formulation #9: (“LT w/o TPA”) 1 μg dual surface/core antigen DNAplasmid vector, 0.5 μg LTA w/o TPA DNA plasmid vector, 0.5 μg LTB w/oTPA DNA plasmid vector;

[0269] Formulation #10: (“LTA”) 1 μg dual surface/core antigen DNAplasmid vector, 1 μg pPJV2004 (LTA) DNA plasmid vector;

[0270] Formulation #11: (“LTA w/o TPA”) 1 μg dual surface/core antigenDNA plasmid vector, 1 μg LTA w/o TPA DNA plasmid vector;

[0271] Formulation #12: (“LTB”) 1 μg dual surface/core antigen DNAplasmid vector, 1 μg pPJV2005 (LTB) DNA plasmid vector; and

[0272] Formulation #13: (“LTB w/o TPA”) 1 μg dual surface/core antigenDNA plasmid vector, 1 μg LTB w/o TPA DNA plasmid vector.

[0273] In a first study, various of the above-described DNA vaccineformulations were administered to five groups of mice using thePowderJect® XR-1 particle delivery device (PowderJect Vaccines Inc.,Madison, Wis.). Each experimental group contained 5 animals, and eachanimal received two immunizations with the respective formulation with afour week resting period between immunizations. The Formulations thatwere tested were as follows: Formulation #1 (no adjuvant); Formulation#2 (CT); Formulation #3 (CT w/o TPA); Formulation #8 (LT); andFormulation #9 (LT w/o TPA). All animals were sacrificed two weeks afterthe second immunization, and the spleens harvested for use in IFN-γ andIL-4 ELISPOT assays. For the cellular immune assays, single cellsuspensions of splenocytes from the spleens of the immunized animalswere cultured in vitro in the presence of a peptide corresponding to aknown T cell epitope (from either the surface or the core antigen) inthe mice. The peptide was dissolved in DMSO (10 mg/ml) and diluted to 10ug/ml in culture.

[0274] For the ELISPOT assays, Millipore Multiscreen membrane filtrationplates were coated with 50 μl of the appropriate antiserum (15 μg/mlanti-IFN-γor-IL-4 antiserum, Pharmingen) in sterile 0.1 M carbonatebuffer (pH 9.6) overnight at 4° C. Plates were washed 6 times withsterile PBS and then blocked with tissue culture medium containing 10%fetal bovine serum (FBS) for 1-2 hr at RT. The medium was removed andspleen cells dispensed into the wells with a total of 1×10⁶ cells perwell. For wells in which less than 1×10⁶ cells from immunized animalswas added, cells from naive animals were used to bring the total to1×10⁶. Cells were incubated overnight in a tissue culture incubator inthe presence of the peptide as described above. The plates were thenwashed 2 time with PBS and 1 time with distilled water. This wasfollowed by 3 washes with PBS. Biotinylated anti-IFN-γ or anti-IL-4monoclonal antibody (Pharmingen) was added to the plate (50 μl of a 1μg/ml solution in PBS) and incubated for 2 hr at RT. The plates werewashed 6 times with PBS after which 50 μl of a Streptavidin Alkalinephosphatase conjugate (1:1000 in PBS, Pharmingen) was added andincubated for 2 hr at RT. The plates were washed 6 times with PBS and analkaline phosphatase color substrate (BioRad) was added and the reactionwas allowed to proceed until dark spots appeared. The reaction wasstopped by washing with water 3 times. Plates were air dried and spotscounted under a microscope.

[0275] The adjuvant effect of the secreted or non-secreted toxin subunitcoding sequences was assessed by determining IFN-γ and IL-4 ELISPOTresponses to both the Hepatitis B surface and core antigens (“sAg” and“cAg”) encoded by the dual surface/core antigen construct, and theseresults were compared as reported in FIGS. 13A-13D. As can be seen, thesecreted (signal sequence containing) CT and LT adjuvant vectors(Formulations #2 and #8, respectively) produced significant increases(P≦0.05) in IFN-γ responses to both the surface and core antigens, andin the IL-4 response to surface antigen (see FIGS. 13A, 13B and 13C).With respect to the IL-4 responses to the core antigen, the lack ofadjuvant effect by the LT vectors (Formulations #8 and #9) is consistentwith observations that the LT toxin is more of a Th1 adjuvant than theCT toxin. Most importantly, the CT and LT vectors lacking the signalsequences (Formulations #3 and #9, respectively) exhibited weakeradjuvant effect, particularly as seen in the IFN-γ ELISPOT data for boththe surface and core antigens (see FIGS. 13A and 13C), where there was astatistically significant drop in adjuvant activity by virtue ofdeletion of the signal sequences.

[0276] Finally, the clearly observable difference in adjuvant effectbetween the secreted (Formulations #2 and #8) and the non-secreted(Formulations #3 and #9) helps establish that the observed adjuvanteffects are not due to CpG motifs within the adjuvant vectors since thesignal-containing and non signal-containing vectors do not have anydifference in bacterial DNA (CpG) content yet exhibit significantdifferences in their ability to augment surface antigen-specific IFN-γresponses.

[0277] In a second study, the above-described DNA vaccine formulationswere administered to eight groups of mice using the PowderJect® XR-1particle delivery device (PowderJect Vaccines Inc., Madison, Wis.). Eachexperimental group contained 5 animals, and each animal received twoimmunizations with the respective formulation with a four week restingperiod between immunizations. The Formulations that were tested were asfollows: Formulation #1 (no adjuvant); Formulation #2 (CT); Formulation#4 (CTA); Formulation #5 (CTA w/o TPA); Formulation #6 (CTB);Formulation #7 (CTB w/o TPA); Formulation #8 (LT); Formulation #10(LTA); Formulation #11 (LTA w/o TPA); Formulation #12 (LTB); andFormulation #13 (LTB w/o TPA). All animals were sacrificed two weeksafter the second immunization, and the spleens harvested for use inIFN-γ and IL-4 ELISPOT assays described herein above.

[0278] As a result of this second study (data not shown), it was againobserved that there was no discernable adjuvant effect that could beattributed to CpG content in the various adjuvant plasmids. Although forthe most part no statistically relevant adjuvant effect was observedwith the various toxin subunit adjuvant vectors, the LT subunit vectors(Formulations #10-13) did show adjuvant effect in the IFN-γ and IL-4response to surface antigen (sAg) that was influenced by thepresence/absence of the secretion signal sequence.

Example 9 Adjuvant Plasmid Vectors Encoding CTA/CTB or LTA/LTB SubunitPeptides in a Viral Challenge Study

[0279] In order to assess the ability of the adjuvant plasmid vectors ofthe present invention to provide for protective effect in a HerpesSimplex Virus type 2 (HSV-2) viral challenge model, the following studywas carried out. A DNA vaccine encoding an HSV-2 antigen was constructedand then combined with various combinations of the present adjuvantplasmid vectors to provide vaccine compositions. After immunization, theimmunized animals were challenged with HSV-2 virus, and the protectiveeffect of the various vaccine compositions was determined.

[0280] With respect to the construction of the DNA antigen plasmid,standard PCR techniques were used to construct the plasmid. The standardPCR conditions that were used for the construction of the vector were asfollows: 1× PCR core buffer with 1.5 mM MgCl₂ (Promega Corp., Madison,Wis.); 0.400 μM of each primer; 200 μM of each dNTP (USB Inc.,Cleveland, Ohio); 2.5 μg Taq polymerase (Promega Corp., Madison, Wis.);1.0 ng template DNA; water to 100 μl; and a mineral oil (AldrichChemical Inc., Milwaukee Wis.) overlay. A PTC-200 thermocycler (MJResearch Inc., Waltham, Mass.) was programmed to run the followingroutine: 4 minutes @ 95° C.; 30 cycles of (1 minute @ 95° C./ 1 minute15 seconds @ 55° C./ 1 minutes @ 72° C.); 10 minutes @ 72° C.; 4° C.hold. The amplification products were removed from the PCR reactionusing the QIAquick® PCR Purification Kit (Qiagen Inc., Valencia Calif.)prior to cutting with restriction enzymes (New England Biolabs, Beverly,Mass.). All PCR products were sequenced after cloning to ensure fidelityof the amplification.

[0281] More specifically, a DNA vaccine plasmid vector encoding theHSV-2 early ICP27 antigen was constructed as follows. HSV is adouble-stranded DNA virus having a genome of about 150-160 kbp. Theviral genome is packaged within an icosahedral nucleocapsid which isenveloped in a membrane. The membrane (or envelope) includes at least 10virus-encoded glycoproteins, the most abundant of which are gB, gC, gD,and gE. The viral genome also encodes over 70 other proteins, includinga group of approximately five ICP antigens. These early proteins aresynthesized early in the viral replication cycle, in contrast to theenvelope glycoproteins which are only made late in the life cycle of thevirus. For a review of the molecular structure and organization of HSV,see, for example, Roizman and Sears (1996) “Herpes simplex viruses andtheir replication” in Fields Virology, 3rd ed., Fields et al. eds.,Lippincott-Raven Publishers, Philadelphia, Pa. The HSV-2 ICP27 antigencan be readily obtained from the HSV-2 genome, for example the genomicregion spanning from approximately nucleotide 114589 to 134980 of theHSV-2 genome, or an EcoRI fragment that spans nucleotides 110931 to139697 of the HSV-2 genome. The sequence of the HSV-2 genome isavailable form published sources, for example the sequence depositedwith GenBank under Accession Number NC_(—)001798.

[0282] In order to construct the ICP27 vector used in the present study,the ICP27 coding region was PCRed from the HSV-2 genome using thefollowing primers: 5′-GCC ACT CTC TTC CGA CAC-3′ (SEQ ID NO:25) and5′-CAA GAA CAT CAC ACG GAA C-3′ (SEQ ID NO:26) to obtain a nucleotidefragment containing nucleotide sequences 114523-116179 (GenBank) ofHSV-2 which correspond to the ICP27 coding region. The ICP27 fragmentwas then cloned into the multiple cloning region of the pTarget vector(Promega Corp., Madison, Wis.).

[0283] The adjuvant plasmid vector constructs containing the CTA, CTB,LTA and LTB toxin subunits (pPJV2002, pPJV2003, pPJV2004, and pPJV2005,respectively) were combined to provide CTA/CTB (pPJV2002+pPJV2003) andLTA/LTB (pPJV2004+pPJV2005) adjuvant. The ICP27 antigen plasmid wascombined with with the toxin subunit vector construct pairs andprecipitated onto 2 micron gold particles. Specifically, plasmid DNA(ICP27 antigen plasmid vector) plus two adjuvant vectors (to provide aCTA/CTB or LTA/LTB combination) was mixed with 2 micron gold particles(Degussa) in a small centrifuge tube containing spermidine.Precipitation was carried out following the methodologies of Example 3,and the coated gold particles were then coated onto the inside surfaceof a TEFZEL® tube as also described in Example 3. The tubing was thencut into 0.5 inch cartridges suitable for loading into the particledelivery device.

[0284] The following DNA-gold formulations were thus generated for theHSV-2 challenge study:

[0285] Formulation #1: (no adjuvant) 2 μg ICP27 antigen DNA plasmidvector;

[0286] Formulation #2: (“High CT”) 900 ng ICP27 antigen DNA plasmidvector, 50 ng of pPJV2002 (CTA), 50 ng of pPJV2003 (CTB);

[0287] Formulation #3: (“Low CT”) 500 ng ICP27 antigen DNA plasmidvector, 250 ng of pPJV2002 (CTA), 250 ng of pPJV2003 (CTB);

[0288] Formulation #4: (“High LT”) 900 ng ICP27 antigen DNA plasmidvector, 50 ng of pPJV2004 (LTA), 50 ng of pPJV2005 (LTB); and

[0289] Formulation #5: (“Low LT”) 500 ng ICP27 antigen DNA plasmidvector, 250 ng of pPJV2004 (LTA), 250 ng of pPJV2005 (LTB).

[0290] In the study, the above-described DNA vaccine formulations wereadministered to five different groups of mice using the PowderJect® XR-1particle delivery device (PowderJect Vaccines Inc., Madison, Wis.). Eachexperimental group contained 12 animals, and each animal received twoimmunizations (single shot applied to the abdomen) with the respectiveformulation with a four week resting period between immunizations. Asixth group of mice was established as a negative (naive) control, anddid not receive any vaccinations. 4 mice from each group were sacked 2weeks after the second immunization and used for IFN-γ ELISPOT assays(data not shown).

[0291] Two weeks post second immunization, all remaining mice (8/group)were challenged with 1×10⁶ PFU of HSV-2 virus, strain MS, viaintra-nasal instillation. The survival graph depicting the results ofthe challenge study is depicted in FIG. 14. As can be seen, 100% of thenaive animals succumbed within 4 days post challenge. The naive animalsare depicted on the graph by the () curve. In addition, 100% of theanimals receiving the ICP27 antigen plasmid vector alone (Formulation#1) died within 7 days post challenge. The animals receiving Formulation#1 are depicted on the graph by the (▾) curve. In marked contrast, the25% (2/8) of the animals receiving the ICP27 plasmid adjuvanted with thelow dose CT (Formulation #3) were protected from the viral challenge,and 38% (3/8) of the animals receiving the ICP27 adjuvanted with thehigh dose CT (Formulation #2) were protected from the viral challenge.The animals receiving Formulation #3 are depicted on the graph by the(▪) curve. The animals receiving Formulation #2 are depicted on thegraph by the (♦) curve. Finally, both the low dose LT-adjuvanted(Formulation #5) and the high dose LT-adjuvanted (Formulation #4) ICP27vaccine provided complete (100%) protection in the immunized animals.The animals receiving Formulation #5 are depicted on the graph by the(▴) curve. The animals receiving Formulation #4 are depicted on thegraph by the (∘) curve.

[0292] Accordingly, novel polynucleotide adjuvant molecules,compositions comprising those adjuvant molecules, and conventional andnucleic acid immunization techniques have been described. Althoughpreferred embodiments of the subject invention have been described insome detail, it is understood that obvious variations can be madewithout departing from the spirit and the scope of the invention asdefined by the appended claims.

1 26 1 5500 DNA pPJV2002 plasmid 1 gacgaaaggg cctcgtgata cgcctatttttataggttaa tgtcatgata ataatggttt 60 cttagacgtc aggtggcact tttcggggaaatgtgcgcgg aacccctatt tgtttatttt 120 tctaaataca ttcaaatatg tatccgctcatgagacaata accctgataa atgcttcaat 180 aatattgaaa aaggaagagt atgagtattcaacatttccg tgtcgccctt attccctttt 240 ttgcggcatt ttgccttcct gtttttgctcacccagaaac gctggtgaaa gtaaaagatg 300 ctgaagatca gttgggtgca cgagtgggttacatcgaact ggatctcaac agcggtaaga 360 tccttgagag ttttcgcccc gaagaacgttttccaatgat gagcactttt aaagttctgc 420 tatgtggcgc ggtattatcc cgtattgacgccgggcaaga gcaactcggt cgccgcatac 480 actattctca gaatgacttg gttgagtactcaccagtcac agaaaagcat cttacggatg 540 gcatgacagt aagagaatta tgcagtgctgccataaccat gagtgataac actgcggcca 600 acttacttct gacaacgatc ggaggaccgaaggagctaac cgcttttttg cacaacatgg 660 gggatcatgt aactcgcctt gatcgttgggaaccggagct gaatgaagcc ataccaaacg 720 acgagcgtga caccacgatg cctgtagcaatggcaacaac gttgcgcaaa ctattaactg 780 gcgaactact tactctagct tcccggcaacaattaataga ctggatggag gcggataaag 840 ttgcaggacc acttctgcgc tcggcccttccggctggctg gtttattgct gataaatctg 900 gagccggtga gcgtgggtct cgcggtatcattgcagcact ggggccagat ggtaagccct 960 cccgtatcgt agttatctac acgacggggagtcaggcaac tatggatgaa cgaaatagac 1020 agatcgctga gataggtgcc tcactgattaagcattggta actgtcagac caagtttact 1080 catatatact ttagattgat ttaaaacttcatttttaatt taaaaggatc taggtgaaga 1140 tcctttttga taatctcatg accaaaatcccttaacgtga gttttcgttc cactgagcgt 1200 cagaccccgt agaaaagatc aaaggatcttcttgagatcc tttttttctg cgcgtaatct 1260 gctgcttgca aacaaaaaaa ccaccgctaccagcggtggt ttgtttgccg gatcaagagc 1320 taccaactct ttttccgaag gtaactggcttcagcagagc gcagatacca aatactgtcc 1380 ttctagtgta gccgtagtta ggccaccacttcaagaactc tgtagcaccg cctacatacc 1440 tcgctctgct aatcctgtta ccagtggctgctgccagtgg cgataagtcg tgtcttaccg 1500 ggttggactc aagacgatag ttaccggataaggcgcagcg gtcgggctga acggggggtt 1560 cgtgcacaca gcccagcttg gagcgaacgacctacaccga actgagatac ctacagcgtg 1620 agcattgaga aagcgccacg cttcccgaagggagaaaggc ggacaggtat ccggtaagcg 1680 gcagggtcgg aacaggagag cgcacgagggagcttccagg gggaaacgcc tggtatcttt 1740 atagtcctgt cgggtttcgc cacctctgacttgagcgtcg atttttgtga tgctcgtcag 1800 gggggcggag cctatggaaa aacgccagcaacgcggcctt tttacggttc ctggcctttt 1860 gctggccttt tgctcacatg ttctttcctgcgttatcccc tgattctgtg gataaccgta 1920 ttaccgcctt tgagtgagct gataccgctcgccgcagccg aacgaccgag cgcagcgagt 1980 cagtgagcga ggaagcggaa gagcgcccaatacgcaaacc gcctctcccc gcgcgttggc 2040 cgattcatta atgcagctgg cacgacaggtttcccgactg gaaagcgggc agtgagcgca 2100 acgcaattaa tgtgagttag ctcactcattaggcacccca ggctttacac tttatgcttc 2160 cggctcgtat gttgtgtgga attgtgagcggataacaatt tcacacagga aacagctatg 2220 accatgatta cgccaagcta gtcgacataaatcaatattg gctattggcc attgcatacg 2280 ttgtatctat atcataatat gtacatttatattggctcat gtccaatatg accgccatgt 2340 tgacattgat tattgactag ttattaatagtaatcaatta cggggtcatt agttcatagc 2400 ccatatatgg agttccgcgt tacataacttacggtaaatg gcccgcctcg tgaccgccca 2460 acgacccccg cccattgacg tcaataatgacgtatgttcc catagtaacg ccaataggga 2520 ctttccattg acgtcaatgg gtggagtatttacggtaaac tgcccacttg gcagtacatc 2580 aagtgtatca tatgccaagt ccggccccctattgacgtca atgacggtaa atggcccgcc 2640 tggcattatg cccagtacat gaccttacgggactttccta cttggcagta catctacgta 2700 ttagtcatcg ctattaccat ggtgatgcggttttggcagt acaccaatgg gcgtggatag 2760 cggtttgact cacggggatt tccaagtctccaccccattg acgtcaatgg gagtttgttt 2820 tggcaccaaa atcaacggga ctttccaaaatgtcgtaata accccgcccc gttgacgcaa 2880 atgggcggta ggcgtgtacg gtgggaggtctatataagca gagctcgttt agtgaaccgt 2940 cagatcgcct ggagacgcca tccacgctgttttgacctcc atagaagaca ccgggaccga 3000 tccagcctcc gcggccggga acggtgcattggaacgcgga ttccccgtgc caagagtgac 3060 gtaagtaccg cctatagact ctataggcacacccctttgg ctcttatgca tgctatactg 3120 tttttggctt ggggcctata cacccccgctccttatgcta taggtgatgg tatagcttag 3180 cctataggtg tgggttattg accattattgaccactcccc tattggtgac gatactttcc 3240 attactaatc cataacatgg ctctttgccacaactatctc tattggctat atgccaatac 3300 tctgtccttc agagactgac acggactctgtatttttaca ggatggggtc ccatttatta 3360 tttacaaatt cacatataca acaacgccgtcccccgtgcc cgcagttttt attaaacata 3420 gcgtgggatc tccacgcgaa tctcgggtacgtgttccgga catgggctct tctccggtag 3480 cggcggagct tccacatccg agccctggtcccatgcctcc agcggctcat ggtcgctcgg 3540 cagctccttg ctcctaacag tggaggccagacttaggcac agcacaatgc ccaccaccac 3600 cagtgtgccg cacaaggccg tggcggtagggtatgtgtct gaaaatgagc tcggagattg 3660 ggctcgcacc gtgacgcaga tggaagacttaaggcagcgg cagaagaaga tgcaggcagc 3720 tgagttgttg tattctgata agagtcagaggtaactcccg ttgcggtgct gttaacggtg 3780 gagggcagtg tagtctgagc agtactcgttgctgccgcgc gcgccaccag acataatagc 3840 tgacagacta acagactgtt cctttccatgggtcttttct gcagtcaccg tccaagcttg 3900 caatcatgga tgcaatgaag agagggctctgctgtgtgct gctgctgtgt ggagcagtct 3960 tcgtttcggc tagcaatgat gataagttatatcgggcaga ttctagacct cctgatgaaa 4020 taaagcagtc aggtggtctt atgccaagaggacagagtga gtactttgac cgaggtactc 4080 aaatgaatat caacctttat gatcatgcaagaggaactca gacgggattt gttaggcacg 4140 atgatggata tgtttccacc tcaattagtttgagaagtgc ccacttagtg ggtcaaacta 4200 tattgtctgg tcattctact tattatatatatgttatagc cactgcaccc aacatgttta 4260 acgttaatga tgtattaggg gcatacagtcctcatccaga tgaacaagaa gtttctgctt 4320 taggtgggat tccatactcc caaatatatggatggtatcg agttcatttt ggggtgcttg 4380 atgaacaatt acatcgtaat aggggctacagagatagata ttacagtaac ttagatattg 4440 ctccagcagc agatggttat ggattggcaggtttccctcc ggagcataga gcttggaggg 4500 aagagccgtg gattcatcat gcaccgccgggttgtgggaa tgctccaaga tcatcgatga 4560 gtaatacttg cgatgaaaaa acccaaagtctaggtgtaaa attccttgac gaataccaat 4620 ctaaagttaa aagacaaata ttttcaggctatcaatctga tattgataca cataatagaa 4680 ttaaggatga attatgagga tcctcgcaatccctaggagg attaggcaag ggcttgagct 4740 cacgctcttg tgagggacag aaatacaatcaggggcagta tatgaatact ccatggagaa 4800 acccagatct acgtatgatc agcctcgactgtgccttcta gttgccagcc atctgttgtt 4860 tgcccctccc ccgtgccttc cttgaccctggaaggtgcca ctcccactgt cctttcctaa 4920 taaaatgagg aaattgcatc gcattgtctgagtaggtgtc attctattct ggggggtggg 4980 gtggggcagg acagcaaggg ggaggattgggaagacaata gcaggcatgc tggggatgcg 5040 gtgggctcta tggcttctga ggcggaaagaaccagctggg gctcgacagc tcgactctag 5100 aattcactgg ccgtcgtttt acaacgtcgtgactgggaaa accctggcgt tacccaactt 5160 aatcgccttg cagcacatcc ccctttcgccagctggcgta atagcgaaga ggcccgcacc 5220 gatcgccctt cccaacagtt gcgcagcctgaatggcgaat ggcgcctgat gcggtatttt 5280 ctccttacgc atctgtgcgg tatttcacaccgcatatggt gcactctcag tacaatctgc 5340 tctgatgccg catagttaag ccagccccgacacccgccaa cacccgctga cgcgccctga 5400 cgggcttgtc tgctcccggc atccgcttacagacaagctg tgaccgtctc cgggagctgc 5460 atgtgtcaga ggttttcacc gtcatcaccgaaacgcgcga 5500 2 5089 DNA pPJV2003 plasmid 2 gacgaaaggg cctcgtgatacgcctatttt tataggttaa tgtcatgata ataatggttt 60 cttagacgtc aggtggcacttttcggggaa atgtgcgcgg aacccctatt tgtttatttt 120 tctaaataca ttcaaatatgtatccgctca tgagacaata accctgataa atgcttcaat 180 aatattgaaa aaggaagagtatgagtattc aacatttccg tgtcgccctt attccctttt 240 ttgcggcatt ttgccttcctgtttttgctc acccagaaac gctggtgaaa gtaaaagatg 300 ctgaagatca gttgggtgcacgagtgggtt acatcgaact ggatctcaac agcggtaaga 360 tccttgagag ttttcgccccgaagaacgtt ttccaatgat gagcactttt aaagttctgc 420 tatgtggcgc ggtattatcccgtattgacg ccgggcaaga gcaactcggt cgccgcatac 480 actattctca gaatgacttggttgagtact caccagtcac agaaaagcat cttacggatg 540 gcatgacagt aagagaattatgcagtgctg ccataaccat gagtgataac actgcggcca 600 acttacttct gacaacgatcggaggaccga aggagctaac cgcttttttg cacaacatgg 660 gggatcatgt aactcgccttgatcgttggg aaccggagct gaatgaagcc ataccaaacg 720 acgagcgtga caccacgatgcctgtagcaa tggcaacaac gttgcgcaaa ctattaactg 780 gcgaactact tactctagcttcccggcaac aattaataga ctggatggag gcggataaag 840 ttgcaggacc acttctgcgctcggcccttc cggctggctg gtttattgct gataaatctg 900 gagccggtga gcgtgggtctcgcggtatca ttgcagcact ggggccagat ggtaagccct 960 cccgtatcgt agttatctacacgacgggga gtcaggcaac tatggatgaa cgaaatagac 1020 agatcgctga gataggtgcctcactgatta agcattggta actgtcagac caagtttact 1080 catatatact ttagattgatttaaaacttc atttttaatt taaaaggatc taggtgaaga 1140 tcctttttga taatctcatgaccaaaatcc cttaacgtga gttttcgttc cactgagcgt 1200 cagaccccgt agaaaagatcaaaggatctt cttgagatcc tttttttctg cgcgtaatct 1260 gctgcttgca aacaaaaaaaccaccgctac cagcggtggt ttgtttgccg gatcaagagc 1320 taccaactct ttttccgaaggtaactggct tcagcagagc gcagatacca aatactgtcc 1380 ttctagtgta gccgtagttaggccaccact tcaagaactc tgtagcaccg cctacatacc 1440 tcgctctgct aatcctgttaccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 1500 ggttggactc aagacgatagttaccggata aggcgcagcg gtcgggctga acggggggtt 1560 cgtgcacaca gcccagcttggagcgaacga cctacaccga actgagatac ctacagcgtg 1620 agcattgaga aagcgccacgcttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 1680 gcagggtcgg aacaggagagcgcacgaggg agcttccagg gggaaacgcc tggtatcttt 1740 atagtcctgt cgggtttcgccacctctgac ttgagcgtcg atttttgtga tgctcgtcag 1800 gggggcggag cctatggaaaaacgccagca acgcggcctt tttacggttc ctggcctttt 1860 gctggccttt tgctcacatgttctttcctg cgttatcccc tgattctgtg gataaccgta 1920 ttaccgcctt tgagtgagctgataccgctc gccgcagccg aacgaccgag cgcagcgagt 1980 cagtgagcga ggaagcggaagagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc 2040 cgattcatta atgcagctggcacgacaggt ttcccgactg gaaagcgggc agtgagcgca 2100 acgcaattaa tgtgagttagctcactcatt aggcacccca ggctttacac tttatgcttc 2160 cggctcgtat gttgtgtggaattgtgagcg gataacaatt tcacacagga aacagctatg 2220 accatgatta cgccaagctagtcgacataa atcaatattg gctattggcc attgcatacg 2280 ttgtatctat atcataatatgtacatttat attggctcat gtccaatatg accgccatgt 2340 tgacattgat tattgactagttattaatag taatcaatta cggggtcatt agttcatagc 2400 ccatatatgg agttccgcgttacataactt acggtaaatg gcccgcctcg tgaccgccca 2460 acgacccccg cccattgacgtcaataatga cgtatgttcc catagtaacg ccaataggga 2520 ctttccattg acgtcaatgggtggagtatt tacggtaaac tgcccacttg gcagtacatc 2580 aagtgtatca tatgccaagtccggccccct attgacgtca atgacggtaa atggcccgcc 2640 tggcattatg cccagtacatgaccttacgg gactttccta cttggcagta catctacgta 2700 ttagtcatcg ctattaccatggtgatgcgg ttttggcagt acaccaatgg gcgtggatag 2760 cggtttgact cacggggatttccaagtctc caccccattg acgtcaatgg gagtttgttt 2820 tggcaccaaa atcaacgggactttccaaaa tgtcgtaata accccgcccc gttgacgcaa 2880 atgggcggta ggcgtgtacggtgggaggtc tatataagca gagctcgttt agtgaaccgt 2940 cagatcgcct ggagacgccatccacgctgt tttgacctcc atagaagaca ccgggaccga 3000 tccagcctcc gcggccgggaacggtgcatt ggaacgcgga ttccccgtgc caagagtgac 3060 gtaagtaccg cctatagactctataggcac acccctttgg ctcttatgca tgctatactg 3120 tttttggctt ggggcctatacacccccgct ccttatgcta taggtgatgg tatagcttag 3180 cctataggtg tgggttattgaccattattg accactcccc tattggtgac gatactttcc 3240 attactaatc cataacatggctctttgcca caactatctc tattggctat atgccaatac 3300 tctgtccttc agagactgacacggactctg tatttttaca ggatggggtc ccatttatta 3360 tttacaaatt cacatatacaacaacgccgt cccccgtgcc cgcagttttt attaaacata 3420 gcgtgggatc tccacgcgaatctcgggtac gtgttccgga catgggctct tctccggtag 3480 cggcggagct tccacatccgagccctggtc ccatgcctcc agcggctcat ggtcgctcgg 3540 cagctccttg ctcctaacagtggaggccag acttaggcac agcacaatgc ccaccaccac 3600 cagtgtgccg cacaaggccgtggcggtagg gtatgtgtct gaaaatgagc tcggagattg 3660 ggctcgcacc gtgacgcagatggaagactt aaggcagcgg cagaagaaga tgcaggcagc 3720 tgagttgttg tattctgataagagtcagag gtaactcccg ttgcggtgct gttaacggtg 3780 gagggcagtg tagtctgagcagtactcgtt gctgccgcgc gcgccaccag acataatagc 3840 tgacagacta acagactgttcctttccatg ggtcttttct gcagtcaccg tccaagcttg 3900 caatcatgga tgcaatgaagagagggctct gctgtgtgct gctgctgtgt ggagcagtct 3960 tcgtttcggc tagcacacctcaaaatatta ctgatttgtg tgcagaatac cacaacacac 4020 aaatatatac gctaaatgataagatatttt cgtatacaga atctctagct ggaaaaagag 4080 agatggctat cattacttttaagaatggtg caatttttca agtagaagta ccaggtagtc 4140 aacatataga ttcacaaaaaaaagcgattg aaaggatgaa ggataccctg aggattgcat 4200 atcttactga agctaaagtcgaaaagttat gtgtatggaa taataaaacg cctcatgcga 4260 ttgccgcaat tagtatggcaaattaaggat cctcgcaatc cctaggagga ttaggcaagg 4320 gcttgagctc acgctcttgtgagggacaga aatacaatca ggggcagtat atgaatactc 4380 catggagaaa cccagatctacgtatgatca gcctcgactg tgccttctag ttgccagcca 4440 tctgttgttt gcccctcccccgtgccttcc ttgaccctgg aaggtgccac tcccactgtc 4500 ctttcctaat aaaatgaggaaattgcatcg cattgtctga gtaggtgtca ttctattctg 4560 gggggtgggg tggggcaggacagcaagggg gaggattggg aagacaatag caggcatgct 4620 ggggatgcgg tgggctctatggcttctgag gcggaaagaa ccagctgggg ctcgacagct 4680 cgactctaga attcactggccgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt 4740 acccaactta atcgccttgcagcacatccc cctttcgcca gctggcgtaa tagcgaagag 4800 gcccgcaccg atcgcccttcccaacagttg cgcagcctga atggcgaatg gcgcctgatg 4860 cggtattttc tccttacgcatctgtgcggt atttcacacc gcatatggtg cactctcagt 4920 acaatctgct ctgatgccgcatagttaagc cagccccgac acccgccaac acccgctgac 4980 gcgccctgac gggcttgtctgctcccggca tccgcttaca gacaagctgt gaccgtctcc 5040 gggagctgca tgtgtcagaggttttcaccg tcatcaccga aacgcgcga 5089 3 5488 DNA pPJV2006 plasmid 3gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 60cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 120tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 180aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 240ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 300ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 360tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 420tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 480actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 540gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 600acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg 660gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 720acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg 780gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag 840ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 900gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct 960cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 1020agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 1080catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 1140tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 1200cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 1260gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 1320taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc 1380ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 1440tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 1500ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 1560cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 1620agcattgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 1680gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 1740atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 1800gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 1860gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 1920ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 1980cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc 2040cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 2100acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc 2160cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg 2220accatgatta cgccaagcta gtcgacataa atcaatattg gctattggcc attgcatacg 2280ttgtatctat atcataatat gtacatttat attggctcat gtccaatatg accgccatgt 2340tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc 2400ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctcg tgaccgccca 2460acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 2520ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 2580aagtgtatca tatgccaagt ccggccccct attgacgtca atgacggtaa atggcccgcc 2640tggcattatg cccagtacat gaccttacgg gactttccta cttggcagta catctacgta 2700ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acaccaatgg gcgtggatag 2760cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt 2820tggcaccaaa atcaacggga ctttccaaaa tgtcgtaata accccgcccc gttgacgcaa 2880atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctcgttt agtgaaccgt 2940cagatcgcct ggagacgcca tccacgctgt tttgacctcc atagaagaca ccgggaccga 3000tccagcctcc gcggccggga acggtgcatt ggaacgcgga ttccccgtgc caagagtgac 3060gtaagtaccg cctatagact ctataggcac acccctttgg ctcttatgca tgctatactg 3120tttttggctt ggggcctata cacccccgct ccttatgcta taggtgatgg tatagcttag 3180cctataggtg tgggttattg accattattg accactcccc tattggtgac gatactttcc 3240attactaatc cataacatgg ctctttgcca caactatctc tattggctat atgccaatac 3300tctgtccttc agagactgac acggactctg tatttttaca ggatggggtc ccatttatta 3360tttacaaatt cacatataca acaacgccgt cccccgtgcc cgcagttttt attaaacata 3420gcgtgggatc tccacgcgaa tctcgggtac gtgttccgga catgggctct tctccggtag 3480cggcggagct tccacatccg agccctggtc ccatgcctcc agcggctcat ggtcgctcgg 3540cagctccttg ctcctaacag tggaggccag acttaggcac agcacaatgc ccaccaccac 3600cagtgtgccg cacaaggccg tggcggtagg gtatgtgtct gaaaatgagc tcggagattg 3660ggctcgcacc gtgacgcaga tggaagactt aaggcagcgg cagaagaaga tgcaggcagc 3720tgagttgttg tattctgata agagtcagag gtaactcccg ttgcggtgct gttaacggtg 3780gagggcagtg tagtctgagc agtactcgtt gctgccgcgc gcgccaccag acataatagc 3840tgacagacta acagactgtt cctttccatg ggtcttttct gcagtcaccg tccaagcttg 3900caatcatgga tgcaatgaag agagggctct gctgtgtgct gctgctgtgt ggagcagtct 3960tcgtttcggc tagcaatgat gataagttat atcgggcaga ttctagacct cctgatgaaa 4020taaagcagtc aggtggtctt atgccaagag gacagagtga gtactttgac cgaggtactc 4080aaatgaatat caacctttat gatcatgcaa gaggaactca gacgggattt gttaggcacg 4140atgatggata tgtttccacc tcaattagtt tgagaagtgc ccacttagtg ggtcaaacta 4200tattgtctgg tcattctact tattatatat atgttatagc cactgcaccc aacatgttta 4260acgttaatga tgtattaggg gcatacagtc ctcatccaga tgaacaagaa gtttctgctt 4320taggtgggat tccatactcc caaatatatg gatggtatcg agttcatttt ggggtgcttg 4380atgaacaatt acatcgtaat aggggctaca gagatagata ttacagtaac ttagatattg 4440ctccagcagc agatggttat ggattggcag gtttccctcc ggagcataga gcttggaggg 4500aagagccgtg gattcatcat gcaccgccgg gttgtgggaa tgctccaaga tcatcgatga 4560gtaatacttg cgatgaaaaa acccaaagtc taggtgtaaa attccttgac gaataccaat 4620ctaaagttaa aagacaaata ttttcaggct atcaatctga tattgataca cataatagaa 4680tttgaggatc ctcgcaatcc ctaggaggat taggcaaggg cttgagctca cgctcttgtg 4740agggacagaa atacaatcag gggcagtata tgaatactcc atggagaaac ccagatctac 4800gtatgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc 4860gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa 4920attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac 4980agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg 5040gcttctgagg cggaaagaac cagctggggc tcgacagctc gactctagaa ttcactggcc 5100gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca 5160gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc 5220caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct ccttacgcat 5280ctgtgcggta tttcacaccg catatggtgc actctcagta caatctgctc tgatgccgca 5340tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg 5400ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg 5460ttttcaccgt catcaccgaa acgcgcga 5488 4 5500 DNA pPJV2004 plasmid 4gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 60cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 120tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 180aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 240ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 300ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 360tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 420tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 480actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 540gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 600acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg 660gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 720acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg 780gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag 840ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 900gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct 960cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 1020agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 1080catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 1140tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 1200cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 1260gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 1320taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc 1380ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 1440tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 1500ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 1560cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 1620agcattgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 1680gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 1740atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 1800gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 1860gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 1920ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 1980cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc 2040cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 2100acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc 2160cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg 2220accatgatta cgccaagcta gtcgacataa atcaatattg gctattggcc attgcatacg 2280ttgtatctat atcataatat gtacatttat attggctcat gtccaatatg accgccatgt 2340tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc 2400ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctcg tgaccgccca 2460acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 2520ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 2580aagtgtatca tatgccaagt ccggccccct attgacgtca atgacggtaa atggcccgcc 2640tggcattatg cccagtacat gaccttacgg gactttccta cttggcagta catctacgta 2700ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acaccaatgg gcgtggatag 2760cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt 2820tggcaccaaa atcaacggga ctttccaaaa tgtcgtaata accccgcccc gttgacgcaa 2880atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctcgttt agtgaaccgt 2940cagatcgcct ggagacgcca tccacgctgt tttgacctcc atagaagaca ccgggaccga 3000tccagcctcc gcggccggga acggtgcatt ggaacgcgga ttccccgtgc caagagtgac 3060gtaagtaccg cctatagact ctataggcac acccctttgg ctcttatgca tgctatactg 3120tttttggctt ggggcctata cacccccgct ccttatgcta taggtgatgg tatagcttag 3180cctataggtg tgggttattg accattattg accactcccc tattggtgac gatactttcc 3240attactaatc cataacatgg ctctttgcca caactatctc tattggctat atgccaatac 3300tctgtccttc agagactgac acggactctg tatttttaca ggatggggtc ccatttatta 3360tttacaaatt cacatataca acaacgccgt cccccgtgcc cgcagttttt attaaacata 3420gcgtgggatc tccacgcgaa tctcgggtac gtgttccgga catgggctct tctccggtag 3480cggcggagct tccacatccg agccctggtc ccatgcctcc agcggctcat ggtcgctcgg 3540cagctccttg ctcctaacag tggaggccag acttaggcac agcacaatgc ccaccaccac 3600cagtgtgccg cacaaggccg tggcggtagg gtatgtgtct gaaaatgagc tcggagattg 3660ggctcgcacc gtgacgcaga tggaagactt aaggcagcgg cagaagaaga tgcaggcagc 3720tgagttgttg tattctgata agagtcagag gtaactcccg ttgcggtgct gttaacggtg 3780gagggcagtg tagtctgagc agtactcgtt gctgccgcgc gcgccaccag acataatagc 3840tgacagacta acagactgtt cctttccatg ggtcttttct gcagtcaccg tccaagcttg 3900caatcatgga tgcaatgaag agagggctct gctgtgtgct gctgctgtgt ggagcagtct 3960tcgtttcggc tagcaatggc gacaaattat accgtgctga ctctagaccc ccagatgaaa 4020taaaacgttc cggaggtctt atgcccagag ggcataatga gtacttcgat agaggaactc 4080aaatgaatat taatctttat gatcacgcga gaggaacaca aaccggcttt gtcagatatg 4140atgacggata tgtttccact tctcttagtt tgagaagtgc tcacttagca ggacagtcta 4200tattatcagg atattccact tactatatat atgttatagc gacagcacca aatatgttta 4260atgttaatga tgtattaggc gtatacagcc ctcacccata tgaacaggag gtttctgcgt 4320taggtggaat accatattct cagatatatg gatggtatcg tgttaatttt ggtgtgattg 4380atgaacgatt acatcgtaac agggaatata gagaccggta ttacagaaat ctgaatatag 4440ctccggcaga ggatggttac agattagcag gtttcccacc ggatcaccaa gcttggagag 4500aagaaccctg gattcatcat gcaccacaag gttgtggaaa ttcatcaaga acaattacag 4560gtgatacttg taatgaggag acccagaatc tgagcacaat atatctcagg aaatatcaat 4620caaaagttaa gaggcagata ttttcagact atcagtcaga ggttgacata tataacagaa 4680ttcgggatga attatgagga tcctcgcaat ccctaggagg attaggcaag ggcttgagct 4740cacgctcttg tgagggacag aaatacaatc aggggcagta tatgaatact ccatggagaa 4800acccagatct acgtatgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt 4860tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa 4920taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg 4980gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg 5040gtgggctcta tggcttctga ggcggaaaga accagctggg gctcgacagc tcgactctag 5100aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt 5160aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc 5220gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt 5280ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag tacaatctgc 5340tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga 5400cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc 5460atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga 5500 5 5089 DNA pPJV2005plasmid 5 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgataataatggttt 60 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatttgtttatttt 120 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataaatgcttcaat 180 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgcccttattccctttt 240 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaagtaaaagatg 300 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaacagcggtaaga 360 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcacttttaaagttctgc 420 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggtcgccgcatac 480 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcatcttacggatg 540 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataacactgcggcca 600 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttgcacaacatgg 660 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagccataccaaacg 720 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaactattaactg 780 gcgaactact tactctagct tcccggcaac aattaataga ctggatggaggcggataaag 840 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgctgataaatctg 900 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagatggtaagccct 960 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaacgaaatagac 1020 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagaccaagtttact 1080 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatctaggtgaaga 1140 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttccactgagcgt 1200 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctgcgcgtaatct 1260 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccggatcaagagc 1320 taccaactct ttttccgaag gtaactggct tcagcagagc gcagataccaaatactgtcc 1380 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccgcctacatacc 1440 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcgtgtcttaccg 1500 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctgaacggggggtt 1560 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatacctacagcgtg 1620 agcattgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtatccggtaagcg 1680 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcctggtatcttt 1740 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtgatgctcgtcag 1800 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttcctggcctttt 1860 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtggataaccgta 1920 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgagcgcagcgagt 1980 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctccccgcgcgttggc 2040 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggcagtgagcgca 2100 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacactttatgcttc 2160 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacaggaaacagctatg 2220 accatgatta cgccaagcta gtcgacataa atcaatattg gctattggccattgcatacg 2280 ttgtatctat atcataatat gtacatttat attggctcat gtccaatatgaccgccatgt 2340 tgacattgat tattgactag ttattaatag taatcaatta cggggtcattagttcatagc 2400 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctcgtgaccgccca 2460 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacgccaataggga 2520 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttggcagtacatc 2580 aagtgtatca tatgccaagt ccggccccct attgacgtca atgacggtaaatggcccgcc 2640 tggcattatg cccagtacat gaccttacgg gactttccta cttggcagtacatctacgta 2700 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acaccaatgggcgtggatag 2760 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgggagtttgttt 2820 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaata accccgccccgttgacgcaa 2880 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctcgtttagtgaaccgt 2940 cagatcgcct ggagacgcca tccacgctgt tttgacctcc atagaagacaccgggaccga 3000 tccagcctcc gcggccggga acggtgcatt ggaacgcgga ttccccgtgccaagagtgac 3060 gtaagtaccg cctatagact ctataggcac acccctttgg ctcttatgcatgctatactg 3120 tttttggctt ggggcctata cacccccgct ccttatgcta taggtgatggtatagcttag 3180 cctataggtg tgggttattg accattattg accactcccc tattggtgacgatactttcc 3240 attactaatc cataacatgg ctctttgcca caactatctc tattggctatatgccaatac 3300 tctgtccttc agagactgac acggactctg tatttttaca ggatggggtcccatttatta 3360 tttacaaatt cacatataca acaacgccgt cccccgtgcc cgcagtttttattaaacata 3420 gcgtgggatc tccacgcgaa tctcgggtac gtgttccgga catgggctcttctccggtag 3480 cggcggagct tccacatccg agccctggtc ccatgcctcc agcggctcatggtcgctcgg 3540 cagctccttg ctcctaacag tggaggccag acttaggcac agcacaatgcccaccaccac 3600 cagtgtgccg cacaaggccg tggcggtagg gtatgtgtct gaaaatgagctcggagattg 3660 ggctcgcacc gtgacgcaga tggaagactt aaggcagcgg cagaagaagatgcaggcagc 3720 tgagttgttg tattctgata agagtcagag gtaactcccg ttgcggtgctgttaacggtg 3780 gagggcagtg tagtctgagc agtactcgtt gctgccgcgc gcgccaccagacataatagc 3840 tgacagacta acagactgtt cctttccatg ggtcttttct gcagtcaccgtccaagcttg 3900 caatcatgga tgcaatgaag agagggctct gctgtgtgct gctgctgtgtggagcagtct 3960 tcgtttcggc tagcgctccc cagtctatta cagaactatg ttcggaatatcgcaacacac 4020 aaatatatac gataaatgac aagatactat catatacgga atcgatggcaggcaaaagag 4080 aaatggttat cattacattt aagagcggcg caacatttca ggtcgaagtcccgggcagtc 4140 aacatataga ctcccaaaaa aaagccattg aaaggatgaa ggacacattaagaatcacat 4200 atctgaccga gaccaaaatt gataaattat gtgtatggaa taataaaacccccaattcaa 4260 ttgcggcaat cagtatggaa aactagggat cctcgcaatc cctaggaggattaggcaagg 4320 gcttgagctc acgctcttgt gagggacaga aatacaatca ggggcagtatatgaatactc 4380 catggagaaa cccagatcta cgtatgatca gcctcgactg tgccttctagttgccagcca 4440 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccactcccactgtc 4500 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtcattctattctg 4560 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatagcaggcatgct 4620 ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctggggctcgacagct 4680 cgactctaga attcactggc cgtcgtttta caacgtcgtg actgggaaaaccctggcgtt 4740 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaatagcgaagag 4800 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatggcgcctgatg 4860 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtgcactctcagt 4920 acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaacacccgctgac 4980 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgtgaccgtctcc 5040 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcga5089 6 5488 DNA pPJV2007 plasmid 6 gacgaaaggg cctcgtgata cgcctatttttataggttaa tgtcatgata ataatggttt 60 cttagacgtc aggtggcact tttcggggaaatgtgcgcgg aacccctatt tgtttatttt 120 tctaaataca ttcaaatatg tatccgctcatgagacaata accctgataa atgcttcaat 180 aatattgaaa aaggaagagt atgagtattcaacatttccg tgtcgccctt attccctttt 240 ttgcggcatt ttgccttcct gtttttgctcacccagaaac gctggtgaaa gtaaaagatg 300 ctgaagatca gttgggtgca cgagtgggttacatcgaact ggatctcaac agcggtaaga 360 tccttgagag ttttcgcccc gaagaacgttttccaatgat gagcactttt aaagttctgc 420 tatgtggcgc ggtattatcc cgtattgacgccgggcaaga gcaactcggt cgccgcatac 480 actattctca gaatgacttg gttgagtactcaccagtcac agaaaagcat cttacggatg 540 gcatgacagt aagagaatta tgcagtgctgccataaccat gagtgataac actgcggcca 600 acttacttct gacaacgatc ggaggaccgaaggagctaac cgcttttttg cacaacatgg 660 gggatcatgt aactcgcctt gatcgttgggaaccggagct gaatgaagcc ataccaaacg 720 acgagcgtga caccacgatg cctgtagcaatggcaacaac gttgcgcaaa ctattaactg 780 gcgaactact tactctagct tcccggcaacaattaataga ctggatggag gcggataaag 840 ttgcaggacc acttctgcgc tcggcccttccggctggctg gtttattgct gataaatctg 900 gagccggtga gcgtgggtct cgcggtatcattgcagcact ggggccagat ggtaagccct 960 cccgtatcgt agttatctac acgacggggagtcaggcaac tatggatgaa cgaaatagac 1020 agatcgctga gataggtgcc tcactgattaagcattggta actgtcagac caagtttact 1080 catatatact ttagattgat ttaaaacttcatttttaatt taaaaggatc taggtgaaga 1140 tcctttttga taatctcatg accaaaatcccttaacgtga gttttcgttc cactgagcgt 1200 cagaccccgt agaaaagatc aaaggatcttcttgagatcc tttttttctg cgcgtaatct 1260 gctgcttgca aacaaaaaaa ccaccgctaccagcggtggt ttgtttgccg gatcaagagc 1320 taccaactct ttttccgaag gtaactggcttcagcagagc gcagatacca aatactgtcc 1380 ttctagtgta gccgtagtta ggccaccacttcaagaactc tgtagcaccg cctacatacc 1440 tcgctctgct aatcctgtta ccagtggctgctgccagtgg cgataagtcg tgtcttaccg 1500 ggttggactc aagacgatag ttaccggataaggcgcagcg gtcgggctga acggggggtt 1560 cgtgcacaca gcccagcttg gagcgaacgacctacaccga actgagatac ctacagcgtg 1620 agcattgaga aagcgccacg cttcccgaagggagaaaggc ggacaggtat ccggtaagcg 1680 gcagggtcgg aacaggagag cgcacgagggagcttccagg gggaaacgcc tggtatcttt 1740 atagtcctgt cgggtttcgc cacctctgacttgagcgtcg atttttgtga tgctcgtcag 1800 gggggcggag cctatggaaa aacgccagcaacgcggcctt tttacggttc ctggcctttt 1860 gctggccttt tgctcacatg ttctttcctgcgttatcccc tgattctgtg gataaccgta 1920 ttaccgcctt tgagtgagct gataccgctcgccgcagccg aacgaccgag cgcagcgagt 1980 cagtgagcga ggaagcggaa gagcgcccaatacgcaaacc gcctctcccc gcgcgttggc 2040 cgattcatta atgcagctgg cacgacaggtttcccgactg gaaagcgggc agtgagcgca 2100 acgcaattaa tgtgagttag ctcactcattaggcacccca ggctttacac tttatgcttc 2160 cggctcgtat gttgtgtgga attgtgagcggataacaatt tcacacagga aacagctatg 2220 accatgatta cgccaagcta gtcgacataaatcaatattg gctattggcc attgcatacg 2280 ttgtatctat atcataatat gtacatttatattggctcat gtccaatatg accgccatgt 2340 tgacattgat tattgactag ttattaatagtaatcaatta cggggtcatt agttcatagc 2400 ccatatatgg agttccgcgt tacataacttacggtaaatg gcccgcctcg tgaccgccca 2460 acgacccccg cccattgacg tcaataatgacgtatgttcc catagtaacg ccaataggga 2520 ctttccattg acgtcaatgg gtggagtatttacggtaaac tgcccacttg gcagtacatc 2580 aagtgtatca tatgccaagt ccggccccctattgacgtca atgacggtaa atggcccgcc 2640 tggcattatg cccagtacat gaccttacgggactttccta cttggcagta catctacgta 2700 ttagtcatcg ctattaccat ggtgatgcggttttggcagt acaccaatgg gcgtggatag 2760 cggtttgact cacggggatt tccaagtctccaccccattg acgtcaatgg gagtttgttt 2820 tggcaccaaa atcaacggga ctttccaaaatgtcgtaata accccgcccc gttgacgcaa 2880 atgggcggta ggcgtgtacg gtgggaggtctatataagca gagctcgttt agtgaaccgt 2940 cagatcgcct ggagacgcca tccacgctgttttgacctcc atagaagaca ccgggaccga 3000 tccagcctcc gcggccggga acggtgcattggaacgcgga ttccccgtgc caagagtgac 3060 gtaagtaccg cctatagact ctataggcacacccctttgg ctcttatgca tgctatactg 3120 tttttggctt ggggcctata cacccccgctccttatgcta taggtgatgg tatagcttag 3180 cctataggtg tgggttattg accattattgaccactcccc tattggtgac gatactttcc 3240 attactaatc cataacatgg ctctttgccacaactatctc tattggctat atgccaatac 3300 tctgtccttc agagactgac acggactctgtatttttaca ggatggggtc ccatttatta 3360 tttacaaatt cacatataca acaacgccgtcccccgtgcc cgcagttttt attaaacata 3420 gcgtgggatc tccacgcgaa tctcgggtacgtgttccgga catgggctct tctccggtag 3480 cggcggagct tccacatccg agccctggtcccatgcctcc agcggctcat ggtcgctcgg 3540 cagctccttg ctcctaacag tggaggccagacttaggcac agcacaatgc ccaccaccac 3600 cagtgtgccg cacaaggccg tggcggtagggtatgtgtct gaaaatgagc tcggagattg 3660 ggctcgcacc gtgacgcaga tggaagacttaaggcagcgg cagaagaaga tgcaggcagc 3720 tgagttgttg tattctgata agagtcagaggtaactcccg ttgcggtgct gttaacggtg 3780 gagggcagtg tagtctgagc agtactcgttgctgccgcgc gcgccaccag acataatagc 3840 tgacagacta acagactgtt cctttccatgggtcttttct gcagtcaccg tccaagcttg 3900 caatcatgga tgcaatgaag agagggctctgctgtgtgct gctgctgtgt ggagcagtct 3960 tcgtttcggc tagcaatggc gacaaattataccgtgctga ctctagaccc ccagatgaaa 4020 taaaacgttc cggaggtctt atgcccagatggcataatga gtacttcgat agaggaactc 4080 aaatgaatat taatctttat gatcacgcgagaggaacaca aaccggcttt gtcagatatg 4140 atgacggata tgtttccact tctcttagtttgagaagtgc tcacttagca ggacagtcta 4200 tattatcagg atattccact tactatatatatgttatagc gacagcacca aatatgttta 4260 atgttaatga tgtattaggc gtatacagccctcacccata tgaacaggag gtttctgcgt 4320 taggtggaat accatattct cagatatatggatggtatcg tgttaatttt ggtgtgattg 4380 atgaacgatt acatcgtaac agggaatatagagaccggta ttacagaaat ctgaatatag 4440 ctccggcaga ggatggttac agattagcaggtttcccacc ggatcaccaa gcttggagag 4500 aagaaccctg gattcatcat gcaccacaaggttgtggaaa ttcatcaaga acaattacag 4560 gtgatacttg taatgaggag acccagaatctgagcacaat atatctcagg aaatatcaat 4620 caaaagttaa gaggcagata ttttcagactatcagtcaga ggttgacata tataacagaa 4680 tttgaggatc ctcgcaatcc ctaggaggattaggcaaggg cttgagctca cgctcttgtg 4740 agggacagaa atacaatcag gggcagtatatgaatactcc atggagaaac ccagatctac 4800 gtatgatcag cctcgactgt gccttctagttgccagccat ctgttgtttg cccctccccc 4860 gtgccttcct tgaccctgga aggtgccactcccactgtcc tttcctaata aaatgaggaa 4920 attgcatcgc attgtctgag taggtgtcattctattctgg ggggtggggt ggggcaggac 4980 agcaaggggg aggattggga agacaatagcaggcatgctg gggatgcggt gggctctatg 5040 gcttctgagg cggaaagaac cagctggggctcgacagctc gactctagaa ttcactggcc 5100 gtcgttttac aacgtcgtga ctgggaaaaccctggcgtta cccaacttaa tcgccttgca 5160 gcacatcccc ctttcgccag ctggcgtaatagcgaagagg cccgcaccga tcgcccttcc 5220 caacagttgc gcagcctgaa tggcgaatggcgcctgatgc ggtattttct ccttacgcat 5280 ctgtgcggta tttcacaccg catatggtgcactctcagta caatctgctc tgatgccgca 5340 tagttaagcc agccccgaca cccgccaacacccgctgacg cgccctgacg ggcttgtctg 5400 ctcccggcat ccgcttacag acaagctgtgaccgtctccg ggagctgcat gtgtcagagg 5460 ttttcaccgt catcaccgaa acgcgcga5488 7 30 DNA synthetic construct 7 ggagctagca atgatgataa gttatatcgg 308 30 DNA synthetic construct 8 cctggatcct cataattcat ccttaattct 30 9 30DNA synthetic construct 9 ggagctagca cacctcaaaa tattactgat 30 10 30 DNAsynthetic construct 10 cctggatcct taatttgcca tactaattgc 30 11 30 DNAsynthetic construct 11 cctggatcct caaattctat tatgtgtatc 30 12 30 DNAsynthetic construct 12 ggagctagca atggcgacaa attataccgt 30 13 30 DNAsynthetic construct 13 cctggatcct cataattcat cccgaattct 30 14 30 DNAsynthetic construct 14 ggagctagcg ctccccagtc tattacagaa 30 15 30 DNAsynthetic construct 15 cctggatccc tagttttcca tactgattgc 30 16 30 DNAsynthetic construct 16 cctggatcct caaattctgt tatatatgtc 30 17 65 DNAsynthetic construct 17 cccaagcttc caccatgagc cttctaaccg aggtcgaaacacctatcaga aacgaatggg 60 agtgc 65 18 28 DNA synthetic construct 18cccggatcct tactccagct ctatgctg 28 19 17 PRT synthetic construct 19 SerLeu Leu Thr Glu Val Glu Thr Pro Ile Arg Asn Glu Trp Glu Cys 1 5 10 15Arg 20 12 PRT synthetic construct 20 Ile Pro Gln Ser Leu Asp Ser Trp TrpThr Ser Leu 1 5 10 21 15 PRT synthetic construct 21 Arg Ile Gln Arg GlyPro Gly Arg Ala Phe Val Ile Thr Gly Lys 1 5 10 15 22 10 PRT syntheticconstruct 22 Arg Gly Pro Gly Arg Ala Phe Val Thr Ile 1 5 10 23 32 DNAsynthetic construct 23 gccgctagca tggacattga cccttataaa ga 32 24 25 DNAsynthetic construct 24 ccaggatcct taacattgag attcc 25 25 18 DNAsynthetic construct 25 gccactctct tccgacac 18 26 19 DNA syntheticconstruct 26 caagaacatc acacggaac 19

What is claimed is:
 1. A composition comprising first and second nucleicacid sequences, wherein said first nucleic acid sequence is a truncatedA subunit coding region obtained or derived from a bacterialADP-ribosylating exotoxin, and said second nucleic acid sequence is atruncated B subunit coding region obtained or derived from a bacterialADP-ribosylating exotoxin, with the proviso that each of said truncatedsubunit coding regions has a 5′ deletion and encodes a subunit peptidenot having an amino terminal bacterial signal peptide.
 2. Thecomposition of claim 1, wherein said first and second nucleic acidsequences are present in a single nucleic acid construct.
 3. Thecomposition of claim 2, wherein said nucleic acid construct is a plasmidvector.
 4. The composition of claim 2, wherein the first and secondnucleic acid sequences are operably linked to a transcriptional controlelement.
 5. The composition of claim 4, wherein said transcriptionalcontrol element is a heterologous promoter.
 6. The composition of claim1 wherein said first and second nucleic acid sequences are present inseparate nucleic acid constructs.
 7. The composition of claim 6, whereinsaid separate nucleic acid constructs are plasmid vectors.
 8. Thecomposition of claim 1, wherein the truncated subunit coding regions areobtained or derived from the same bacterial ADP-ribosylating exotoxin.9. The composition of claim 8, wherein said bacterial ADP-ribosylatingexotoxin is a cholera toxin (CT).
 10. The composition of claim 8,wherein said bacterial ADP-ribosylating exotoxin is an E. coli heatlabile enterotoxin (LT).
 11. The composition of claim 1, wherein atleast one of the truncated subunit coding regions has been geneticallymodified to detoxify the subunit peptide encoded thereby.
 12. Thecomposition of claim 11, wherein the truncated A subunit coding regionhas been genetically modified to disrupt or inactivate ADP-ribosyltransferase activity in the subunit peptide encoded thereby.
 13. Thecomposition of claim 1, wherein the truncated A subunit coding regionhas been further genetically modified so as to delete a C-terminal KDELor RDEL motif in the subunit peptide encoded thereby.
 14. Thecomposition of claim 1 further comprising an antigen of interest. 15.The composition of claim 14, wherein said antigen is from a bacterial,viral or parasitic pathogen.
 16. The composition of claim 1, furthercomprising a third nucleic acid sequence that encodes an antigen ofinterest.
 17. The composition of claim 16, wherein said antigen is froma bacterial, viral or parasitic pathogen.
 18. The composition of claim16, wherein said third nucleic acid sequence is present in a nucleicacid construct that does not contain said first or said second nucleicacid sequence.
 19. The composition of claim 18, wherein the nucleic acidconstruct containing the third nucleic acid sequence is a plasmidvector.
 20. The composition of claim 16, wherein said third nucleic acidsequence is present in a nucleic acid construct that also contains atleast one of said first or said second nucleic acid sequence.
 21. Thecomposition of claim 20, wherein the nucleic acid construct containingthe third nucleic acid sequence is a plasmid vector.
 22. The compositionof claim 1, wherein said composition is in a particulate form.
 23. Thecomposition of claim 22, wherein said particulate composition issuitable for transdermal delivery via a particle delivery device. 24.The composition of claim 1, further comprising a pharmaceuticallyacceptable vehicle or excipient.
 25. The composition of claim 1, whereinthe first and second nucleic acid sequences are coated onto a corecarrier particle.
 26. The composition of claim 25, wherein the corecarrier particle has an average diameter of about 0.1 to about 10 μm.27. The composition of claim 25, wherein the core carrier particlecomprises a metal.
 28. The composition of claim 27 wherein the metal isgold.
 29. The composition of claim 28 wherein the core carrier particlehas a diameter of about 1 to about 3 μm.
 30. The composition of claim 1further comprising a transfection facilitating agent.
 31. Thecomposition of claim 30, wherein the transfection facilitating agent isa liposome.
 32. A composition comprising first and second nucleic acidsequences, wherein said first nucleic acid sequence is a modified Asubunit coding region obtained or derived from a bacterialADP-ribosylating exotoxin, and said second nucleic acid sequence is a Bsubunit coding region obtained or derived from a bacterialADP-ribosylating exotoxin, with the proviso that said modified A subunitcoding region and said B subunit coding region each encode a maturesubunit peptide, and with the further proviso that the modified Asubunit coding region has been genetically modified so as to delete aC-terminal KDEL or RDEL motif in the subunit peptide encoded thereby.33. The composition of claim 32, wherein said first and second nucleicacid sequences are present in a single nucleic acid construct.
 34. Thecomposition of claim 33, wherein said nucleic acid construct is aplasmid vector.
 35. The composition of claim 33, wherein the first andsecond nucleic acid sequences are operably linked to a transcriptionalcontrol element.
 36. The composition of claim 35, wherein saidtranscriptional control element is a heterologous promoter.
 37. Thecomposition of claim 32, wherein said first and second nucleic acidsequences are present in separate nucleic acid constructs.
 38. Thecomposition of claim 37, wherein said separate nucleic acid constructsare plasmid vectors.
 39. The composition of claim 32, wherein the B andmodified A subunit coding regions are obtained or derived from the samebacterial ADP-ribosylating exotoxin.
 40. The composition of claim 39,wherein said bacterial ADP-ribosylating exotoxin is a cholera toxin(CT).
 41. The composition of claim 39, wherein said bacterialADP-ribosylating exotoxin is an E. coli heat labile enterotoxin (LT).42. The composition of claim 32, wherein at least one of the B ormodified A subunit coding regions has been genetically modified todetoxify the subunit peptide encoded thereby.
 43. The composition ofclaim 42, wherein the modified A subunit coding region has beengenetically modified to disrupt or inactivate ADP-ribosyl transferaseactivity in the subunit peptide encoded thereby.
 44. The composition ofclaim 32, wherein the modified A subunit coding region and the B subunitcoding region have each been truncated by a 5′ deletion whereby each ofsaid truncated subunit coding regions encodes a subunit peptide nothaving an amino terminal bacterial signal peptide.
 45. The compositionof claim 32 further comprising an antigen of interest.
 46. Thecomposition of claim 45, wherein said antigen is from a bacterial, viralor parasitic pathogen.
 47. The composition of claim 32 furthercomprising a third nucleic acid sequence that encodes an antigen ofinterest.
 48. The composition of claim 47, wherein said antigen is froma bacterial, viral or parasitic pathogen.
 49. The composition of claim47, wherein said third nucleic acid sequence is present in a nucleicacid construct that does not contain said first or said second nucleicacid sequence.
 50. The composition of claim 49, wherein the nucleic acidconstruct containing the third nucleic acid sequence is a plasmidvector.
 51. The composition of claim 47, wherein said third nucleic acidsequence is present in a nucleic acid construct that also contains atleast one of said first or said second nucleic acid sequence.
 52. Thecomposition of claim 51, wherein the nucleic acid construct containingthe third nucleic acid sequence is a plasmid vector.
 53. The compositionof claim 32, wherein said composition is in a particulate form.
 54. Thecomposition of claim 53, wherein said particulate composition issuitable for transdermal delivery via a particle delivery device. 55.The composition of claim 32 further comprising a pharmaceuticallyacceptable vehicle or excipient.
 56. A composition according to claim55, wherein the first and second nucleic acid sequences are coated ontoa core carrier particle.
 57. The composition of claim 56, wherein thecore carrier particle has an average diameter of about 0.1 to about 10μm.
 58. The composition of claim 56, wherein the core carrier particlecomprises a metal.
 59. The composition of claim 58, wherein the metal isgold.
 60. The composition of claim 59 wherein the core carrier particlehas a diameter of about 1 to about 3 μm.
 61. The composition of claim 32further comprising a transfection facilitating agent.
 62. Thecomposition of claim 61, wherein the transfection facilitating agent isa liposome.
 63. A method for enhancing an immune response against anantigen of interest in a vertebrate subject, the method comprising: (a)administering the antigen of interest to the subject; (b) providing anadjuvant composition comprising first and second nucleic acid sequences,wherein said first nucleic acid sequence is a truncated A subunit codingregion obtained or derived from a bacterial ADP-ribosylating exotoxin,and said second nucleic acid sequence is a truncated B subunit codingregion obtained or derived from a bacterial ADP-ribosylating exotoxin,with the proviso that each of said truncated subunit coding regions hasa 5′ deletion and encodes a subunit peptide not having an amino terminalbacterial signal peptide; and (c) administering said adjuvantcomposition to the subject, whereby upon introduction to the subject,the first and second nucleic acid sequences are expressed to providesubunit peptides in an amount sufficient to elicit said enhanced immuneresponse against the antigen of interest.
 64. The method of claim 63,wherein the antigen of interest and the adjuvant composition areadministered to the same site in the subject.
 65. The method of claim63, wherein the antigen of interest and the adjuvant composition areadministered concurrently.
 66. The method of claim 65, wherein theantigen of interest and the adjuvant composition are combined to providea single vaccine composition.
 67. The method of claim 63, wherein theantigen of interest is from a bacterial, viral or parasitic pathogen.68. The method of claim 67, wherein step (a) entails administering athird nucleic acid sequence that encodes said antigen of interest. 69.The method of claim 63, wherein the adjuvant composition is administeredto the subject in particulate form.
 70. The method of claim 69, whereinsaid first and second nucleic acid sequences are coated onto a corecarrier particle and administered to the subject using aparticle-mediated delivery technique.
 71. The method of claim 63,wherein the subject is a mammal.
 72. A method for enhancing an immuneresponse against an antigen of interest in a vertebrate subject, themethod comprising: (a) administering the antigen of interest to thesubject; (b) providing an adjuvant composition comprising first andsecond nucleic acid sequences, wherein said first nucleic acid sequenceis a modified A subunit coding region obtained or derived from abacterial ADP-ribosylating exotoxin, and said second nucleic acidsequence is a B subunit coding region obtained or derived from abacterial ADP-ribosylating exotoxin, with the proviso that said modifiedA subunit coding region and said B subunit coding region each encode amature subunit peptide, and with the further proviso that the modified Asubunit coding region has been genetically modified so as to delete aC-terminal KDEL or RDEL motif in the subunit peptide encoded thereby;and (c) administering said adjuvant composition to the subject, wherebyupon introduction to the subject, the first and second nucleic acidsequences are expressed to provide subunit peptides in an amountsufficient to elicit said enhanced immune response against the antigenof interest.
 73. The method of claim 72, wherein the antigen of interestand the adjuvant composition are administered to the same site in thesubject.
 74. The method of claim 72, wherein the antigen of interest andthe adjuvant composition are administered concurrently.
 75. The methodof claim 74, wherein the antigen of interest and the adjuvantcomposition are combined to provide a single vaccine composition. 76.The method of claim 72, wherein the antigen of interest is from abacterial, viral or parasitic pathogen.
 77. The method of claim 76,wherein step (a) entails administering a third nucleic acid sequencethat encodes said antigen of interest.
 78. The method of claim 72,wherein the adjuvant composition is administered to the subject inparticulate form.
 79. The method of claim 78, wherein said first andsecond nucleic acid sequences are coated onto a core carrier particleand administered to the subject using a particle-mediated deliverytechnique.
 80. The method of 72, wherein the subject is a mammal.